Mannich base prodrugs of 3-(pyrrol-2-ylmethylidene)-2-indolinone derivatives

ABSTRACT

The present invention is directed to Mannich base prodrugs of certain 3-(pyrrol-2-ylmethylidene)-2-indolinone derivatives that modulate the activity of protein kinases (“PKs”). Pharmaceutical compositions comprising these compounds, methods of treating diseases related to abnormal PK activity utilizing pharmaceutical compositions comprising these compounds and methods of preparing them are also disclosed.

CROSS-REFERENCE

This application claims priority under 35 U.S.C. 119(e) to U.S.Provisional applications Ser. No. 60/207,000 filed on May 24, 2000, andSer. No. 60/225,045, filed on Aug. 11, 2000, the disclosures of whichare incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention is directed to Mannich base prodrugs of certain3-(pyrrol-2-ylmethylidene)-2-indolinone derivatives that modulate theactivity of protein kinases (“PKs”). Pharmaceutical compositionscomprising these compounds, methods of treating diseases related toabnormal PK activity utilizing pharmaceutical compositions comprisingthese compounds and methods of preparing them are also disclosed.

2. State of the Art

Protein kinases (“PKs”) are enzymes that catalyze the phosphorylation ofhydroxy groups on tyrosine, serine and threonine residues of proteins.PKs can be conveniently broken down into two classes, the proteintyrosine kinases (PTKs) and the serine-threonine kinases (STKs). One ofthe prime aspects of PTK activity is their involvement with growthfactor receptors. Growth factor receptors are cell-surface proteins.When bound by a growth factor ligand, growth factor receptors areconverted to an active form which interacts with proteins on the innersurface of a cell membrane. This leads to phosphorylation on tyrosineresidues of the receptor and other proteins and to the formation insidethe cell of complexes with a variety of cytoplasmic signaling moleculesthat, in turn, effect numerous cellular responses such as cell division(proliferation), cell differentiation, cell growth, expression ofmetabolic effects to the extracellular microenvironment, etc (See.,Schlessinger and Ullrich (1992) Neuron 9:303-391).

Growth factor receptors with PTK activity are known as receptor tyrosinekinases (“RTKs”). They comprise a large family of transmembranereceptors with diverse biological activity. At present, at leastnineteen (19) distinct subfamilies of RTKs have been identified. Anexample of these is the subfamily designated the “HER” RTKs, whichinclude EGFR (epithelial growth factor receptor), HER2, HER3 and HER4.

Another RTK subfamily consists of insulin receptor (IR), insulin-likegrowth factor I receptor (IGF-1R) and insulin receptor related receptor(IRR). IR and IGF-1R interact with insulin, IGF-I and IGF-II to form aheterotetramer of two entirely extracellular glycosylated α subunits andtwo β subunits which cross the cell membrane and which contain thetyrosine kinase domain.

A third RTK subfamily is referred to as the platelet derived growthfactor receptor (“PDGFR”) group, which includes PDGFRα, PDGFRβ, CSFIR,c-kit and c-fins. Another group is the fetus liver kinase (“flk”)receptor subfamily. This group is believed to be made of up of kinaseinsert domain-receptor fetal liver kinase-1 (KDR/FLK-1), flk-1R, flk-4and fins-like tyrosine kinase 1 (flt-1).

A further member of the tyrosine kinase growth factor receptor family isthe fibroblast growth factor (“FGF”) receptor subgroup. This groupconsists of four receptors, FGFR1-4, and seven ligands, FGF1-7. Whilenot yet well defined, it appears that the receptors consist of aglycosylated extracellular domain containing a variable number ofimmunoglobin-like loops and an intracellular domain in which thetyrosine kinase sequence is interrupted by regions of unrelated aminoacid sequences.

Still another member of the tyrosine kinase growth factor receptorfamily is the vascular endothelial growth factor (“VEGF”) receptorsubgroup. VEGF is a dimeric glycoprotein similar to PDGF but hasdifferent biological functions and target cell specificity in vivo. Inparticular, VEGF is presently thought to play an essential role isvasculogenesis and angiogenesis.

A more complete listing of the known RTK subfamilies is described inPlowman et al., DN&P, 7(6):334-339 (1994) which is incorporated byreference, including any drawings, as if fully set forth herein.

In addition to the RTKs, there also exists a family of entirelyintracellular PTKs called “non-receptor tyrosine kinases” or “cellulartyrosine kinases.” This latter designation, abbreviated “CTK,” will beused herein. CTKs do not contain extracellular and transmembranedomains. At present, over 24 CTKs in 11 subfamilies (Src, Frk, Btk, Csk,Abl, Zap70, Fes, Fps, Fak, Jak and Ack) have been identified. The Srcsubfamily appear so far to be the largest group of CTKs and includesSrc, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. For a more detaileddiscussion of CTKs, see Bolen, Oncogene, 8:2025-2031 (1993), which isincorporated by reference, including any drawings, as if fully set forthherein.

The serine/threonine kinases, STKs, like the CTKs, are predominantlyintracellular although there are a few receptor kinases of the STK type.STKs are the most common of the cytosolic kinases; i.e., kinases thatperform their function in that part of the cytoplasm other than thecytoplasmic organelles and cytoskelton. The cytosol is the region withinthe cell where much of the cell's intermediary metabolic andbiosynthetic activity occurs; e.g., it is in the cytosol that proteinsare synthesized on ribosomes.

RTKs, CTKs and STKs have all been implicated in a host of pathogenicconditions including, significantly, cancer. Other pathogenic conditionswhich have been associated with PTKs include, without limitation,psoriasis, hepatic cirrhosis, diabetes, angiogenesis, restenosis, oculardiseases, rheumatoid arthritis and other inflammatory disorders,immunological disorders such as autoimmune disease, cardiovasculardisease such as atherosclerosis and a variety of renal disorders.

With regard to cancer, two of the major hypotheses advanced to explainthe excessive cellular proliferation that drives tumor developmentrelate to functions known to be PK regulated. That is, it has beensuggested that malignant cell growth results from a breakdown in themechanisms that control cell division and/or differentiation. It hasbeen shown that the protein products of a number of proto-oncogenes areinvolved in the signal transduction pathways that regulate cell growthand differentiation. These protein products of proto-oncogenes includethe extracellular growth factors, transmembrane growth factor PTKreceptors (RTKs), cytoplasmic PTKs (CTKs) and cytosolic STKs, discussedabove.

In view of the apparent link between PK-related cellular activities andwide variety of human disorders, it is no surprise that a great deal ofeffort is being expended in an attempt to identify ways to modulate PKactivity. For example, attempts have been made to identify smallmolecules which act as PK inhibitors. For example, bis-monocylic,bicyclic and heterocyclic aryl compounds (PCT WO 92/20642),vinylene-azaindole derivatives (PCT WO 94/14808) and1-cyclopropyl-4-pyridylquinolones (U.S. Pat. No. 5,330,992) have beendescribed as tyrosine kinase inhibitors. Styryl compounds (U.S. Pat. No.5,217,999), styryl-substituted pyridyl compounds (U.S. Pat. No.5,302,606), quinazoline derivatives (EP Application No. 0 566 266 A1),selenaindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxyliccompounds (PCT WO 92/21660), and benzylphosphonic acid compounds (PCT WO91/15495). Additionally, a family of novel pyrrole-substituted2-indolinone compounds have been discovered which exhibit PK modulatingability and have a salutary effect against disorders related to abnormalPK activity (U.S. Pat. No. 5,792,783 and PCT Application Publication No.WO 99/61422). Administration of various species of pyrrole-substituted2-indolinone compounds has been shown to be an effective therapeuticapproach to cure many kinds of solid tumors. For example,3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one, ahighly active selective inhibitor of the vascular endothelial growthfactor receptor (Flk-1/KDR), inhibits tyrosine kinase catalysis, tumorvascularization, and growth of multiple tumor types (Fong et al. (1999)Cancer Res. 59:99-106). These compounds, however, have highlipophilicity and low solubility in water and most common vehicles atphysiological pH limit their formulation and hence their administration.

Accordingly, there is a need for PK inhibitors that do not exhibit suchdrawbacks. The present invention fulfills this and related needs.

SUMMARY OF THE INVENTION

In one aspect, this invention is directed to a compound of Formula (I):

wherein:

R³, R⁴, R⁵ and R⁶ are independently selected from the group consistingof hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto,alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido,trihalomethane-sulfonamido, carbonyl, C-carboxy, O-carboxy, C-amido,N-amido, cyano, nitro, halo, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, amino and —NR¹¹R¹² where R¹¹ and R¹² are independentlyselected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl,carbonyl, acetyl, sulfonyl, and trifluoromethanesulfonyl, or R¹¹ andR¹², together with the nitrogen atom to which they are attached, combineto form a five- or six-member heteroalicyclic ring provided that atleast two of R³, R⁴, R⁵ and R⁶ are hydrogen; or

R³ and R⁴, R⁴ and R⁵, or R⁵ and R⁶ combine to form a six-member arylring, a methylenedioxy or an ethylenedioxy group;

R⁷ is selected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,aryloxy, carbonyl, acetyl, C-amido, C-thioamido, amidino, C-carboxy,O-carboxy, sulfonyl, and trihalomethane-sulfonyl;

R⁸, R⁹ and R¹⁰ are independently selected from the group consisting ofhydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto,alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido,carbonyl, C-carboxy, O-carboxy, cyano, nitro, halo, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino and—NR¹¹R¹², wherein R¹¹ and R¹² are as defined above;

R^(1′) is hydrogen or alkyl; and

R^(3′) and R^(4′) are independently alkyl, or R^(3′) and R^(4′),together with the nitrogen atom to which they are attached, combine toform a heteroalicyclic ring or a heteroaryl ring provided that theheteroalicyclic ring is not piperidin-1-yl or morpholin-4-yl; or apharmaceutically acceptable salt thereof.

Specifically, the compounds of the present invention convert in vivo tocompounds of Formula (II):

that exhibit PK modulating ability, in particular PK inhibiting ability,and are therefore useful in treating disorders related to abnormal PKactivity. The active compounds (II) formed from the compounds of thepresent invention are described in U.S. Pat. No. 5,792,783, PCTApplication Publication No. WO 99/61422, and U.S. patent applicationSer. No. 09/783,264, filed on Feb. 15, 2001, and titled “PYRROLESUBSTITUTED 2-INDOLINONE AS PROTEIN KINASE INHIBITORS”, the disclosuresof which are hereby incorporated by reference.

The prodrug compounds of the present invention have advantages overcompounds of Formula (II) by virtue of improved aqueous solubility andformulability. For example, Applicants have discovered that theN-pyrrolidin-1-ylmethyl prodrug of compound (II) where R³-R⁷ and R⁹ arehydrogen and R⁸ and R⁹ are methyl provides unexpected increased aqueoussolubility over the parent compound thus making it particularly suitablefor IV formulations. It is contemplated that similar enhanced solubilitywill be observed for other claimed compounds of Formula (I) withN-pyrrolidin-1-ylmethyl moiety or other —CHR^(1′)NR^(3′)R^(4′) groupsthat are within the scope of the present invention. A generaldescription of the advantages and uses of prodrugs as pharmaceuticallyuseful compounds is given in an article by Walter and George in Br. J.Clin. Pharmac., Vol. 28, pp. 497-507, 1989.

In a second aspect this invention is directed to a pharmaceuticalcomposition comprising one or more compound(s) of Formula (I) or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable excipient.

In a third aspect, this invention is directed to a method of treatingdiseases mediated by abnormal protein kinase activity, in, particular,receptor tyrosine kinases (RTKs), non-receptor protein tyrosine kinases(CTKs) and serine/threonine protein kinases (STKs), in an organism, inparticular humans, which method comprises administering to said organisma pharmaceutical composition comprising a compound of Formula (I), apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable excipient. Such diseases include by way of example and notlimitation, cancer, diabetes, hepatic cirrhosis, cardiovascular diseasesuch as atherosclerosis, angiogenesis, immunological disease such asautoimmune disease e.g., AIDS and lupus and renal disease. Specifically,the diseases mediated by EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFRα,PDGFRβ, CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R,FGFR-2R, FGFR-3R, FGFR-4R, Src, Frk, Btk, Csk, Abl, ZAP70, Fes/Fps, Fak,Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr, Yrk, CDK2 and Raf.

In a fourth aspect, this invention is directed to a method of modulatingthe catalytic activity (e.g., inhibiting the catalytic activity) of PKs,in particular receptor tyrosine kinases (RTKs), non-receptor proteintyrosine kinases (CTKs) and serine/threonine protein kinases (STKs),using a compound of this invention or a pharmaceutical compositioncomprising a compound of this invention and a pharmaceuticallyacceptable excipient. The method may be carried out in vitro or in vivo.In particular, the receptor protein kinase whose catalytic activity ismodulated by a compound of this invention is selected from the groupconsisting of EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFRα, PDGFRβ,CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R, FGFR-2R,FGFR-3R and FGFR-4R. The cellular tyrosine kinase whose catalyticactivity is modulated by a compound of this invention is selected fromthe group consisting of Src, Frk, Btk, Csk, Abl, ZAP70, Fes/Fps, Fak,Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. Theserine-threonine protein kinase whose catalytic activity is modulated bya compound of this invention is selected from the group consisting ofCDK2 and Raf.

In a fifth aspect, this invention is directed to the use of a compoundof Formula (I) in the preparation of a medicament useful in thetreatment of a disease mediated by abnormal PK activity.

In a sixth aspect, this invention is directed to a method of preparing acompound of Formula (I) which method comprises reacting a compound ofFormula (II)

with an amine of formula —NR^(3′)R^(4′) in the presence of an aldehydeof formula R^(1′)CHO where R^(1′), R^(3′) and R^(4′) are as defined inFormula (I) above;

optionally modifying any of the R³-R¹⁰ groups;

optionally preparing an acid addition salt.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise stated the following terms used in the specificationand claims have the meanings discussed below:

“Alkyl” refers to a saturated aliphatic hydrocarbon including straightchain, or branched chain groups. Preferably, the alkyl group has 1 to 20carbon atoms (whenever a numerical range; e.g., “1-20”, is statedherein, it means that the group, in this case the alkyl group, maycontain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. up to andincluding 20 carbon atoms). More preferably, it is a medium size alkylhaving 1 to 10 carbon atoms. Most preferably, it is a lower alkyl having1 to 4 carbon atoms e.g., methyl, ethyl, n-propyl, isopropyl, butyl,iso-butyl, tert-butyl and the like. The alkyl group may be substitutedor unsubstituted. When substituted, the substituent group(s) ispreferably one or more, more preferably one or two groups, individuallyselected from the group consisting of cycloalkyl, aryl, heteroaryl,heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio,arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy,nitro, silyl, amino, ammonium and —NR¹³R¹⁴ where R¹³ and R¹⁴ areindependently selected from the group consisting of hydrogen, alkyl,unsubstituted alkyl, cycloalkyl, aryl, carbonyl, acetyl, sulfonyl,amino, and trifluoromethanesulfonyl, or R¹³ and R¹⁴, together with thenitrogen atom to which they are attached, combine to form a five- orsix-member heteroalicyclic ring. More preferably, the substituent ishydroxy, amino, or —NR¹³R¹⁴ where R¹³ and R¹⁴ are independently selectedfrom the group consisting of unsubstituted lower alkyl, lower alkylsubstituted with amino or hydroxy, or R¹³ and R¹⁴, together with thenitrogen atom to which they are attached, combine to form pyrrolidine,morpholine, or piperazine.

A “cycloalkyl” group refers to an all-carbon monocyclic ring (i.e.,rings which share an adjacent pair of carbon atoms) of 3 to 6 ring atomswherein one of more of the rings does not have a completely conjugatedpi-electron system e.g, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, and the like.Examples, without limitation, of cycloalkyl groups are cyclopropane,cyclobutane, cyclopentane, cyclopentene, cyclohexane, adamantane,cyclohexadiene, cycloheptane and, cycloheptatriene. A cycloalkyl groupmay be substituted or unsubstituted. When substituted, the substituentgroup(s) is preferably one or more, more preferably one or two groups,individually selected from alkyl, unsubstituted alkyl, aryl, heteroaryl,heteroalicyclic, unsubstituted heteroalicyclic, hydroxy, alkoxy,aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl,thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl, C-amido,N-amido, nitro, amino and —NR¹³R¹⁴, with R¹³ and R¹⁴ as defined above.

An “alkenyl” group refers to an alkyl group, as defined herein,consisting of at least two carbon atoms and at least one carbon-carbondouble bond e.g., ethenyl, propenyl, butenyl or pentenyl and theirstructural isomeric forms such as 1- or 2-propenyl, 1-, 2-, or 3-butenyland the like.

An “alkynyl” group refers to an alkyl group, as defined herein,consisting of at least two carbon atoms and at least one carbon-carbontriple bond e.g., acetylene, ethnyl, propynyl, butynyl, or pentnyl andtheir structural isomeric forms as described above.

An “aryl” group refers to an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups of 6 to 12 ring atoms and having a completely conjugatedpi-electron system. Examples, without limitation, of aryl groups arephenyl, naphthalenyl and anthracenyl. The aryl group may be substitutedor unsubstituted. When substituted, the substituted group(s) ispreferably one or more, more preferably one, two, or three substituents,independently selected from the group consisting of halo, trihalomethyl,alkyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano,nitro, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl,sulfonyl, amino and —NR¹³R¹⁴, with R¹³ and R¹⁴ as defined above.Preferably the substituent(s) is/are independently selected from chloro,fluoro, bromo, methyl, ethyl, propyl including all its isomeric forms,butyl including all its isomeric forms, hydroxy, methoxy, phenoxy, thio,methylthio, phenylthio, cyano, nitro, carboxy, methoxycarbonyl, oramino.

A “heteroaryl” group refers to a monocyclic or fused aromatic ring(i.e., rings which share an adjacent pair of atoms) of 5 to 9 ring atomsin which one, two, three or four ring atoms are selected from the groupconsisting of nitrogen, oxygen and sulfur and the rest being carbon.Examples, without limitation, of heteroaryl groups are pyrrole, furan,thiophene, imidazole, oxazole, thiazole, pyrazole, tetrazole, pyridine,pyrimidine, quinoline, isoquinoline, purine and carbazole. Theheteroaryl group may be substituted or unsubstituted. When substituted,the substituted group(s) is preferably one or more, more preferably oneor two substituents, independently selected from the group consisting ofalkyl, cycloalkyl, halo, trihalomethyl, hydroxy, alkoxy, aryloxy,mercapto, alkylthio, arylthio, cyano, nitro, carbonyl, thiocarbonyl,sulfonamido, C-carboxy, O-carboxy, sulfinyl, sulfonyl, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino and—NR¹³R¹⁴, with R¹³ and R¹⁴ as defined above. Preferably thesubstituent(s) is/are independently selected from the group consistingof chloro, fluoro, bromo, methyl, ethyl, propyl including all itsisomeric forms, butyl including all its isomeric forms, hydroxy,methoxy, phenoxy, thio, methylthio, phenylthio, cyano, nitro, carboxy,methoxycarbonyl, or amino.

A “heteroalicyclic” group refers to a monocyclic or fused ring of 4 to 9ring atoms containing one, two, or three heteroatoms in the ring whichare selected from the group consisting of nitrogen, oxygen and —S(O)_(n)where n is 0-2, the remaining ring atoms being carbon. The rings mayalso have one or more double bonds. However, the rings do not have acompletely conjugated pi-electron system. Examples, without limitation,of heteroalicyclic groups are pyrrolidine, piperidine, piperazine,morpholine, imidazolidine, tetrahydropyridazine, tetrahydrofuran,thiomorpholine, tetrahydropyridine, and the like. The heteroalicyclicring may be substituted or unsubstituted. When substituted, thesubstituted group(s) is preferably one or more, more preferably one,two, or three substituents, independently selected from the groupconsisting of alkyl, cycloalkyl, halo, trihalomethyl, hydroxy, alkoxy,aryloxy, mercapto, alkylthio, arylthio, cyano, nitro, carbonyl,thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-arbamyl,O-thiocarbamyl, N-thiocarbamyl, sulfinyl, sulfonyl, C-amido, N-amido,amino and —NR¹³R¹⁴, with R¹³ and R¹⁴ as defined above. Preferably thegroup(s) is/are selected from the group consisting of chloro, fluoro,bromo, methyl, ethyl, propyl including all its isomeric forms, butylincluding all its isomeric forms, hydroxy, methoxy, phenoxy, thio,methylthio, phenylthio, cyano, nitro, carboxy, methoxycarbonyl, oramino.

A “hydroxy” group refers to an —OH group.

An “alkoxy” group refers to an —O-unsubstituted alkyl, —O-substitutedalkyl and an —O-unsubstitutedcycloalkyl group, as defined herein.Examples include and are not limited to methoxy, ethoxy, propoxy,butoxy, cyclopropyloxy, and the like, preferably methoxy.

An “aryloxy” group refers to both an —O-aryl and an —O-heteroaryl group,as defined herein. Examples include and are not limited to phenoxy,napthyloxy, pyridyloxy, furanyloxy, and the like.

A “mercapto” group refers to an —SH group.

A “alkylthio” group refers to both an S-alkyl and an —S-cycloalkylgroup, as defined herein. Examples include and are not limited tomethylthio, ethylthio, and the like.

A “arylthio” group refers to both an —S-aryl and an —S-heteroaryl group,as defined herein. Examples include and are not limited to phenylthio,napthylthio, pyridylthio, furanylthio, and the like.

A “sulfinyl” group refers to a —S(═O)—R″ group wherein, in addition tobeing as defined below, R″ may also be a hydroxy group, e.g.,methylsulfinyl, phenylsulfinyl, and the like.

A “sulfonyl” group refers to a —S(═O)₂R″ group wherein, in addition tobeing as defined below, R″ may also be a hydroxy group e.g.,methylsulfonyl, ethylsulfonyl, phenylsulfonyl, and the like.

A “trihalomethyl” group refers to a —CX₃ group wherein X is a halo groupas defined herein e.g., trifluoromethyl, trichloromethyl,tribromomethyl, dichlorofluoromethyl, and the like.

A “trihalomethanesulfonyl” group refers to a X₃CS(═O)₂— groups with X asdefined above, e.g., trifluoromethylsulfonyl, trichloromethylsulfonyl,tribromomethylsulfonyl, and the like.

A “trihalomethanesulfonylamido” group refers to a —NH—S(═O)₂R groupswherein R is trihalomethyl as defined above.

“Carbonyl” and “acyl” are used interchangeably herein to refer to a—C(═O)—R″ group, where R″ is selected from the group consisting ofhydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ringcarbon) and heteroalicyclic (bonded through a ring carbon), as definedherein. Representative examples include and the not limited to acetyl,propionyl, benzoyl, formyl, cyclopropylcarbonyl, pyridinylcarbonyl,pyrrolidin-1-ylcarbonyl, and the like

An “aldehyde” group refers to a carbonyl group where R″ is hydrogen.

A “thiocarbonyl” group refers to a —C(═S)—R″ group, with R″ as definedherein.

A “C-carboxy” group refers to a —C(═O)O—R″ group, with R″ as definedherein e.g., —COOH, methoxycarbonyl, ethoxycarbonyl, benzyloxycarbonyl,and the like.

An “O-carboxy” group refers to a —OC(═O)R″ group, with R″ as definedherein e.g., methylcarbonyloxy, phenylcarbonyloxy, benzylcarbonyloxy,and the like.

An “ester” group refers to a —C(═O)O—R″ group with R″ as defined hereinexcept that R″ cannot be hydrogen e.g., methoxycarbonyl,benzyloxycarbonyl, and the like.

An “acetyl” group refers to a —C(═O)CH₃ group.

A “carboxylic acid” group refers to a C-carboxy group in which R″ ishydrogen.

A “halo” group refers to fluorine, chlorine, bromine or iodine.

A “cyano” group refers to a —C≡N group.

A “nitro” group refers to a —NO₂ group.

A “methylenedioxy” group refers to —OCH₂O— group where the two oxygenatoms are bonded to adjacent carbon atoms.

An “ethylenedioxy” group refers to —OCH₂CH₂O— where the two oxygen atomsare bonded to adjacent carbon atoms.

An “S-sulfonamido” group refers to a —S(═O)₂NR¹³R¹⁴ group, with R¹³ andR¹⁴ as defined herein. Representative examples include and are notlimited to dimethylaminosulfonyl, aminosulfonyl,phenylmethylaminosulfonyl, phenylaminosulfonyl, and the like.

An “N-sulfonamido” group refers to a —NR¹³S(═O)₂R¹⁴ group, with R¹³ andR¹⁴ as defined herein e.g., methylsulfonylamino, ethylsulfonylamino,phenylsulfonylamino, benzylsulfonylamino, and the like.

An “O-carbamyl” group refers to a —OC(═O)NR¹³R¹⁴ group with R¹³ and R¹⁴as defined herein.

An “N-carbamyl” group refers to a R¹⁴OC(═O)NR¹³— group, with R¹³ and R¹⁴as defined herein.

An “O-thiocarbamyl” group refers to a —OC(═S)NR¹³R¹⁴ group with R¹³ andR¹⁴ as defined herein.

An “N-thiocarbamyl” group refers to a R¹⁴OC(═S)NR¹³— group, with R¹³ andR¹⁴ as defined herein.

An “amino” group refers to an —NR¹³R¹⁴ group, wherein R¹³ and R¹⁴ areindependently hydrogen or unsubstituted lower alkyl e.g, —NH₂,dimethylamino, diethylamino, ethylamino, methylamino, and the like.

A “C-amido” group refers to a —C(═O)NR¹³R¹⁴ group with R¹³ and R¹⁴ asdefined herein. Preferably R¹³ is hydrogen or unsubstituted lower alkyland R¹⁴ is hydrogen, lower alkyl optionally substituted withheteroalicyclic, hydroxy, or amino. For example, —C(═O)NR¹³R¹⁴ may beaminocarbonyl, dimethylaminocarbonyl, diethylaminocarbonyl,diethylaminoethylaminocarbonyl, ethylaminoethylaminocarbonyl,2-morpholinoethylaminocarbonyl, 3-morpholinopropylaminocarbonyl,3-morpholino-2-hydroxypropylaminocarbonyl, and the like.

An “N-amido” group refers to a R¹⁴C(═O)NR¹³— group, with R¹³ and R¹⁴ asdefined herein e.g., acetylamino, and the like.

A “ammonium” group refers to a —⁺NR¹⁵R¹⁶R¹⁷ group, wherein R¹⁵ and R¹⁶are independently selected from the group consisting of alkyl,cycloalkyl, aryl, and heteroaryl, and R¹⁷ is selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aryl, and heteroaryl.

A “amidino” group refers to a R¹⁵R¹⁶NC(═NR¹⁷)— group, with R¹⁵, R¹⁶ andR¹⁷ as defined herein.

A “morpholino” group refers to a group having the chemical structure

A “piperazinyl” group refers to a group having the chemical structure:

The terms “indolinone”, “2-indolinone” and “indolin-2-one” are usedinterchangeably herein to refer to a molecule having the chemicalstructure:

“Pyrrole” refers to a molecule having the chemical structure:

“Pyrrole-substituted 2-indolinone” and “3-pyrrol-1-yl-2-indolinone” areused interchangeably herein to refer to a chemical compound having thegeneral structure shown in Formula II.

A “prodrug” refers to an agent which is converted into the parent drugin vivo. Prodrugs are often useful because, in some situations, they maybe easier to administer than the parent drug. They may, for instance, bebioavailable by oral administration whereas the parent drug is not. Theprodrug may also have improved solubility in pharmaceutical compositionsover the parent drug. A prodrug may be converted into the parent drug byvarious mechanisms, including enzymatic processes and metabolichydrolysis. See Harper, “Drug Latentiation” in Jucker, ed. Progress inDrug Research 4:221-294 (1962); Morozowich et al., “Application ofPhysical Organic Principles to Prodrug Design” in E. B. Roche ed. Designof Biopharmaceutical Properties through Prodrugs and Analogs, APHA Acad.Pharm. Sci. (1977); Bioreversible Carriers in Drug in Drug Design,Theory and Application, E. B. Roche, ed., APHA Acad. Pharm. Sci. (1987);Design of Prodrugs, H. Bundgaard, Elsevier (1985); Wang et al. “Prodrugapproaches to the improved delivery of peptide drug” in Curr. Pharm.Design. 5(4):265-287 (1999); Pauletti et al. (1997) Improvement inpeptide bioavailabilty: Peptidomimetics and Prodrug Strategies, Adv.Drug. Delivery Rev. 27:235-256; Mizen et al. (1998) “The Use of Estersas Prodrugs for Oral Delivery of β-Lactam antibiotics,” Pharm. Biotech.11,:345-365; Gaignault et al. (1996) “Designing Prodrugs andBioprecursors I. Carrier Prodrugs,” Pract. Med. Chem. 671-696;Asgharnejad, “Improving Oral Drug Transport”, in Transport Processes inPharmaceutical Systems, G. L. Amidon, P. I. Lee and E. M. Topp, Eds.,Marcell Dekker, p. 185-218 (2000); Balant et al., “Prodrugs for theimprovement of drug absorption via different routes of administration”,Eur. J Drug Metab. Pharmacokinet., 15(2): 143-53 (1990); Balimane andSinko, “Involvement of multiple transporters in the oral absorption ofnucleoside analogues”, Adv. Drug Delivery Rev., 39(1-3): 183-209 (1999);Browne, “Fosphenytoin (Cerebyx)”, Clin. Neuropharmacol. 20(1): 1-12(1997); Bundgaard, “Bioreversible derivatization of drugs—principle andapplicability to improve the therapeutic effects of drugs”, Arch. Pharm.Chemi 86(1): 1-39 (1979); Bundgaard H. “Improved drug delivery by theprodrug approach”, Controlled Drug Delivery 17: 179-96 (1987); BundgaardH. “Prodrugs as a means to improve the delivery of peptide drugs”, Adv.Drug Delivery Rev. 8(1): 1-38 (1992); Fleisher et al. “Improved oraldrug delivery: solubility limitations overcome by the use of prodrugs”,Adv. Drug Delivery Rev. 19(2): 115-130 (1996); Fleisher et al. “Designof prodrugs for improved gastrointestinal absorption by intestinalenzyme targeting”, Methods Enzymol. 112 (Drug Enzyme Targeting, Pt. A):360-81, (1985); Farquhar D, et al., “Biologically ReversiblePhosphate-Protective Groups”, J. Pharm. Sci., 72(3): 324-325 (1983);Freeman S, et al., “Bioreversible Protection for the Phospho Group:Chemical Stability and Bioactivation of Di(4-acetoxy-benzyl)Methylphosphonate with Carboxyesterase,” J. Chem. Soc., Chem. Commun.,875-877 (1991); Friis and Bundgaard, “Prodrugs of phosphates andphosphonates: Novel lipophilic alpha-acyloxyalkyl ester derivatives ofphosphate- or phosphonate containing drugs masking the negative chargesof these groups”, Eur. J Pharm. Sci. 4: 49-59 (1996); Gangwar et al.,“Pro-drug, molecular structure and percutaneous delivery”, Des.Biopharm. Prop. Prodrugs Analogs, [Symp.] Meeting Date 1976, 409-21.(1977); Nathwani and Wood, “Penicillins: a current review of theirclinical pharmacology and therapeutic use”, Drugs 45(6): 866-94 (1993);Sinhababu and Thakker, “Prodrugs of anticancer agents”, Adv. DrugDelivery Rev. 19(2): 241-273 (1996); Stella et al., “Prodrugs. Do theyhave advantages in clinical practice?”, Drugs 29(5): 455-73 (1985); Tanet al. “Development and optimization of anti-HIV nucleoside analogs andprodrugs: A review of their cellular pharmacology, structure-activityrelationships and pharmacokinetics”, Adv. Drug Delivery Rev. 39(1-3):117-151 (1999); Taylor, “Improved passive oral drug delivery viaprodrugs”, Adv. Drug Delivery Rev., 19(2): 131-148 (1996); Valentino andBorchardt, “Prodrug strategies to enhance the intestinal absorption ofpeptides”, Drug Discovery Today 2(4): 148-155 (1997); Wiebe and Knaus,“Concepts for the design of anti-HIV nucleoside prodrugs for treatingcephalic HIV infection”, Adv. Drug Delivery Rev.: 39(1-3):63-80 (1999);Waller et al., “Prodrugs”, Br. J. Clin. Pharmac. 28: 497-507 (1989).

The compounds of this invention may possess one or more chiral centers,and can therefore be produced as individual stereoisomers or as mixturesof stereoisomers, depending on whether individual stereoisomers ormixtures of stereoisomers of the starting materials are used. Unlessindicated otherwise, the description or naming of a compound or group ofcompounds is intended to include both the individual stereoisomers ormixtures (racemic or otherwise) of stereoisomers. Methods for thedetermination of stereochemistry and the separation of stereoisomers arewell known to a person of ordinary skill in the art [see the discussionin Chapter 4 of March J: Advanced Organic Chemistry, 4th ed. John Wileyand Sons, New York, N.Y., 1992].

The chemical formulae referred to herein may exhibit the phenomena oftautomerism and structural isomerism. For example, the compoundsdescribed herein may adopt an E or a Z configuration about the doublebond connecting the 2-indolinone moiety to the pyrrole moiety or theymay be a mixture of E and Z. This invention encompasses any tautomericor structural isomeric form and mixtures thereof.

The term “method” refers to manners, means, techniques and proceduresfor accomplishing a given task including, but not limited to, thosemanners, means, techniques and procedures either known to, or readilydeveloped from known manners, means, techniques and procedures by,practitioners of the chemical, pharmaceutical, biological, biochemicaland medical arts.

As used herein, the term “modulation” or “modulating” refers to thealteration of the catalytic activity of RTKs, CTKs and STKs. Inparticular, modulating refers to the activation of the catalyticactivity of RTKs, CTKs and STKs, preferably the activation or inhibitionof the catalytic activity of RTKs, CTKs and STKs, depending on theconcentration of the compound or salt to which the RTK, CTK or STK isexposed or, more preferably, the inhibition of the catalytic activity ofRTKs, CTKs and STKs.

The term “catalytic activity” as used herein refers to the rate ofphosphorylation of tyrosine under the influence, direct or indirect, ofRTKs and/or CTKs or the phosphorylation of serine and threonine underthe influence, direct or indirect, of STKs.

The term “contacting” as used herein refers to bringing a compound ofthis invention and a target PK together in such a manner that thecompound can affect the catalytic activity of the PK, either directly,i.e., by interacting with the kinase itself, or indirectly, i.e., byinteracting with another molecule on which the catalytic activity of thekinase is dependent. Such “contacting” can be accomplished in vitro,i.e., in a test tube, a petri dish or the like. In a test tube,contacting may involve only a compound and a PK of interest or it mayinvolve whole cells. Cells may also be maintained or grown in cellculture dishes and contacted with a compound in that environment. Inthis context, the ability of a particular compound to affect a PKrelated disorder, i.e., the IC₅₀ of the compound, defined below, can bedetermined before use of the compounds in vivo with more complex livingorganisms is attempted. For cells outside the organism, multiple methodsexist, and are well-known to those skilled in the art, to get the PKs incontact with the compounds including, but not limited to, direct cellmicroinjection and numerous transmembrane carrier techniques.

“In vitro” refers to procedures performed in an artificial environmentsuch as, e.g., without limitation, in a test tube or culture medium. Theskilled artisan will understand that, for example, an isolated PK may becontacted with a modulator in an in vitro environment. Alternatively, anisolated cell may be contacted with a modulator in an in vitroenvironment.

As used herein, “in vivo” refers to procedures performed within a livingorganism such as, without limitation, a mouse, rat, rabbit, ungulate,bovine, equine, porcine, canine, feline, primate, or human.

As used herein, “PK related disorder,” “PK driven disorder,” and“abnormal PK activity” all refer to a condition characterized byinappropriate, i.e., under or, more commonly, over PK catalyticactivity, where the particular PK can be an RTK, a CTK or an STK.Inappropriate catalytic activity can arise as the result of either: (1)PK expression in cells which normally do not express PKs, (2) increasedPK expression leading to unwanted cell proliferation, differentiationand/or growth, or, (3) decreased PK expression leading to unwantedreductions in cell proliferation, differentiation and/or growth.Over-activity of a PK refers to either amplification of the geneencoding a particular PK or production of a level of PK activity whichcan correlate with a cell proliferation, differentiation and/or growthdisorder (that is, as the level of the PK increases, the severity of oneor more of the symptoms of the cellular disorder increases).Under-activity is, of course, the converse, wherein the severity of oneor more symptoms of a cellular disorder increase as the level of the PKactivity decreases.

The term “organism” refers to any living entity comprised of at leastone cell. A living organism can be as simple as, for example, a singleeukaryotic cell or as complex as a mammal, including a human being.

The term “therapeutically effective amount” as used herein refers tothat amount of the compound being administered which will relieve tosome extent one or more of the symptoms of the disorder being treated.In reference to the treatment of cancer, a therapeutically effectiveamount refers to that amount which has the effect of (1) reducing thesize of the tumor, (2) inhibiting (that is, slowing to some extent,preferably stopping) tumor metastasis, (3) inhibiting to some extent(that is, slowing to some extent, preferably stopping) tumor growth,and/or, (4) relieving to some extent (or, preferably, eliminating) oneor more symptoms associated with the cancer.

“Pharmaceutically acceptable salt” refers to those salts which retainthe biological effectiveness and properties of the free bases and whichare obtained by reaction with inorganic or organic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, malic acid, citric acid, maleicacid, succinic acid, tartaric acid, and the like.

A “pharmaceutical composition” refers to a mixture of one or more of thecompounds described herein, or a pharmaceutically acceptable saltsthereof, with other chemical components, such as physiologicallyacceptable carriers and excipients. The purpose of a pharmaceuticalcomposition is to facilitate administration of a compound to anorganism.

As used herein, a “pharmaceutically acceptable carrier” refers to acarrier or diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound.

An “excipient” refers to an inert substance added to a pharmaceuticalcomposition to further facilitate administration of a compound.Examples, without limitation, of excipients include calcium carbonate,calcium phosphate, various sugars and types of starch, cellulosederivatives, gelatin, vegetable oils and polyethylene glycols.

“Treating” or “treatment” of a disease includes preventing the diseasefrom occurring in an animal that may be predisposed to the disease butdoes not yet experience or exhibit symptoms of the disease (prophylactictreatment), inhibiting the disease (slowing or arresting itsdevelopment), providing relief from the symptoms or side-effects of thedisease (including palliative treatment), and relieving the disease(causing regression of the disease). With regard to cancer, these termssimply mean that the life expectancy of an individual affected with acancer will be increased or that one or more of the symptoms of thedisease will be reduced.

By “monitoring” is meant observing or detecting the effect of contactinga compound with a cell expressing a particular PK. The observed ordetected effect can be a change in cell phenotype, in the catalyticactivity of a PK or a change in the interaction of a PK with a naturalbinding partner. Techniques for observing or detecting such effects arewell-known in the art. For example, the catalytic activity of a PK maybe observed by determining the rate or amount of phosphorylation of atarget molecule. The above-referenced effect is selected from a changeor an absence of change in a cell phenotype, a change or absence ofchange in the catalytic activity of said protein kinase or a change orabsence of change in the interaction of said protein kinase with anatural binding partner in a final aspect of this invention.

“Cell phenotype” refers to the outward appearance of a cell or tissue orthe biological function of the cell or tissue. Examples, withoutlimitation, of a cell phenotype are cell size, cell growth, cellproliferation, cell differentiation, cell survival, apoptosis, andnutrient uptake and use. Such phenotypic characteristics are measurableby techniques well-known in the art.

A “natural binding partner” refers to a polypeptide that binds to aparticular PK in a cell. Natural binding partners can play a role inpropagating a signal in a PK-mediated signal transduction process. Achange in the interaction of the natural binding partner with the PK canmanifest itself as an increased or decreased concentration of thePK/natural binding partner complex and, as a result, in an observablechange in the ability of the PK to mediate signal transduction.

PRESENTLY PREFERRED COMPOUNDS

While the broadest definition of the invention is set out in the Summaryof the Invention, certain compounds of this invention are presentlypreferred.

Presently preferred compounds of this invention are compounds of Formula(I) where:

R³, R⁵, and R⁶ are independently selected from the group consisting ofhydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto,alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido,trihalomethane-sulfonamido, carbonyl, C-carboxy, O-carboxy, C-amido,N-amido, cyano, nitro, halo, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, amino, and —NRR¹¹R¹² where R¹¹ and R¹² are independentlyselected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl,carbonyl, acetyl, sulfonyl, trifluoromethanesulfonyl or, R¹¹ and R¹²,together with the nitrogen to which they are attached, combined form afive- or six-member heteroalicyclic ring; especially R³, R⁵, and R⁶ arehydrogen;

R⁷ is hydrogen;

R⁴ is hydrogen or halo, especially hydrogen, fluoro, or chloro,particularly hydrogen or fluoro;

R^(1′) is hydrogen or methyl, especially hydrogen;

R⁸ and R¹⁰ are independently unsubstituted lower alkyl, especiallymethyl;

R⁹ is hydrogen, lower alkyl substituted with C-carboxy or —C(═O)NHR¹²wherein R¹² is lower alkyl substituted with amino or heteroalicyclic andoptionally substituted with hydroxy; R⁹ is most preferably hydrogen,3-carboxypropyl, (2-diethylaminoethyl)-aminocarbonyl,(2-ethylaminoethyl)aminocarbonyl,3-(morpholin-4-yl)propyl-aminocarbonyl,3-(morpholin-4-yl)-2-hydroxypropylaminocarbonyl, particularly hydrogen,3-carboxypropyl, (2-diethylaminoethyl)aminocarbonyl, or(2-ethylaminoethyl)-aminocarbonyl; and

R^(3′) and R^(4′) are lower alkyl optionally substituted hydroxy,especially methyl, ethyl, 2-hydroxyethyl; or R^(3′) and R^(4′), togetherwith the nitrogen atom to which they are attached, form pyrrolidin-1-yl,2-(S)-hydroxymethylpyrrolidin-1-yl, 2-(S)-carboxy-pyrrolidin-1-yl,piperazin-1-yl, or 4-methylpiperazin-1-yl, especially pyrrolidin-1-yl;or

R³′ and R^(4′) together with the nitrogen atom to which they areattached form a heteroaryl ring, preferably, pyrro-1-yl, pyridin-1-yl,oxazol-3-yl, isoxazol-2-yl, pyrazin-1-yl, pyradizin-1-yl, quinolin-1-yl,imidazol-1-yl, more preferably pyridin-1-yl.

A number of different preferences have been given above, and followingany one of these preferences results in a compound of this inventionthat is more presently preferred than a compound in which thatparticular preference is not followed. However, these preferences aregenerally independent [although some (alternative) preferences aremutually exclusive], and additive; and following more than one of thesepreferences may result in a more presently preferred compound than onein which fewer of the preferences are followed.

Presently preferred classes of compounds of this invention include thosewhere:

(a) R^(1′), R³, R⁴, R⁵, R⁶, R⁷, and R⁹ are hydrogen; R⁸ and R¹⁰ areunsubstituted lower alkyl, especially methyl; and R^(3′) and R^(4′),together with the nitrogen atom to which they are attached, formpyrrolidin-1-yl, 2-(S)-hydroxymethylpyrrolidin-1-yl,2-(S)-carboxypyrrolidin-1-yl, piperazin-1-yl, or 4-methylpiperazin-1-yl,especially pyrrolidin-1-yl.

(b) R^(1′), R³, R⁴, R⁵, R⁶, and R⁷ are hydrogen; R⁸ and R¹⁰ areunsubstituted lower alkyl, especially methyl; R⁹ is lower alkylsubstituted with C-carboxy, especially 3-carboxypropyl; and R^(3′) andR^(4′), together with the nitrogen atom to which they are attached formpyrrolidin-1-yl, 2-(S)-hydroxymethylpyrrolidin-1-yl,2-(S)-carboxy-pyrrolidin-1-yl, piperazin-1-yl, or4-methylpiperazin-1-yl, especially pyrrolidin-1-yl.

(c) R^(1′), R³, R⁵, R⁶, and R⁷ are hydrogen; R⁴ is halo, especiallyfluoro, R⁸ and R¹⁰ are unsubstituted lower alkyl, especially methyl; R⁹is —C(═O)NHR¹³ wherein R¹³ is lower alkyl substituted with amino orheteroalicyclic and optionally substituted with hydroxy, especially(2-diethylaminoethyl)-aminocarbonyl, (2-ethylaminoethyl)-aminocarbonyl,3-(morpholin-4-yl)propylaminocarbonyl,3-(morpholin-4-yl)-2-hydroxypropylaminocarbonyl, particularly(2-diethylaminoethyl)aminocarbonyl, or(2-ethylaminoethyl)-aminocarbonyl; and R^(3′) and R^(4′), together withthe nitrogen atom to which they are attached, form pyrrolidin-1-yl,2-(S)-hydroxymethylpyrrolidin-1-yl, 2-(S)-carboxypyrrolidin-1-yl,piperazin-1-yl, or 4-methylpiperazin-1-yl, especially pyrrolidin-1-yl.

(d) R^(1′), R³, R⁴, R⁵, R⁶, R⁷, and R⁹ are hydrogen; R⁹ and R¹⁰ areunsubstituted lower alkyl, especially methyl; and R^(3′) and R^(4′),together with the nitrogen atom to which they are attached, form aheteroaryl ring, preferably, pyrrol-1-yl, pyridin-1-yl, oxazol-3-yl,isoxazol-2-yl, pyrazin-1-yl, pyridazin-1-yl, quinolin-1-yl,imidazol-1-yl, more preferably pyridin-1-yl.

Presently preferred compounds of this invention include:

(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one;

(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(4-methylpiperazin-1-ylmethyl)-1,3-dihydro-2H-indol-2-one;

(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-[2(S)-hydroxymethyl-1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one;

(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-[2(S)-carboxy-1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one.

(3Z)-3-{[3,5-dimethyl-4-(2-diethylaminoethylaminocarbonyl)-1H-pyrrol-2-yl]-methylidene}-1-(1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one;

(3Z)-3-{[3,5-dimethyl-4-(2-ethylaminoethylaminocarbonyl)-1H-pyrrol-2-yl]-methylidene}-1-(1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one;and

(3Z)-3-{[3,5-dimethyl-4-(3-morpholin-4-yl-2-hydroxypropylaminocarbonyl)-1H-pyrrol-2-yl]-methylidene}-1-(1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one.

GENERAL SYNTHETIC SCHEME

The starting materials and reagents used in preparing these compoundsare either available from commercial suppliers such as Aldrich ChemicalCo., (Milwaukee, Wis.), Bachem (Torrance, Calif.), or Sigma (St. Louis,Mo.) or are prepared by methods known to those skilled in the artfollowing procedures set forth in references such as Fieser and Fieser'sReagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons,1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition) and Larock's ComprehensiveOrganic Transformations (VCH Publishers Inc., 1989). These schemes aremerely illustrative of some methods by which the compounds of thisinvention can be synthesized, and various modifications to these schemescan be made and will be suggested to one skilled in the art havingreferred to this disclosure. The starting materials and theintermediates of the reaction may be isolated and purified if desiredusing conventional techniques, including but not limited to filtration,distillation, crystallization, chromatography and the like. Suchmaterials may be characterized using conventional means, includingphysical constants and spectral data. Unless specified to the contrary,the reactions described herein take place at atmospheric pressure over atemperature range from about −78° C. to about 150° C., more preferablyfrom about 0° C. to about 125° C. and most preferably at about room (orambient) temperature, e.g., about 20° C.

Compounds of Formula (I) where R^(3′) and R^(4′) are independently alkylor combine to form a heteroalicyclic ring may be prepared as illustratedand described below:

A compound of Formula (I) where R³-R¹⁰ and R^(1′), R^(3′) and R^(4′) areas described in the Summary of the Invention can be prepared by reactinga compound of formula (II) with an aldehyde such as formaldehyde,acetaldehyde, and the like, and a suitable amine.

The solvent in which the reaction is carried out may be a protic or anaprotic solvent, preferably it is a protic solvent such as an alcohole.g., methanol or ethanol, or an aqueous alcohol. The reaction may becarried out at temperatures greater than room temperature. Thetemperature is generally from about 20° C. to about 100° C., preferablyabout 40° C. to about 80° C. By “about” is meant that the temperaturerange is preferably within 10 degrees Celsius of the indicatedtemperature, more preferably within 5 degrees Celsius of the indicatedtemperature and, most preferably, within 2 degrees Celsius of theindicated temperature. Thus, for example, by “about 60° C.” is meant 60°C.±10° C., preferably 60° C.±5° C. and most preferably, 60° C.±2° C.

Suitable amines include alicyclic and cyclic secondary amines. Theseamines are either commercially available from Aldrich, Sigma, etc., orthey can be prepared by methods well known in the art. Exemplarysecondary amines include dimethylamine, diethylamine andbis(2-hydroxyethyl)amine. Exemplary cyclic secondary amines includeN-alkyl piperazine, pyrrolidine, proline, 2-hydroxymethylpyrrolidine,3,5-dimethylpiperazine, 2-methylpyrrolidine, and morpholine.

Compounds of Formula (I), where R^(3′) and R^(4′) combine to form aheteroaryl ring, may be prepared by reacting the parent3-pyrrolidinyl-2-indolinone (II) with a suitable aldehyde to yield anintermediate N-hydroxyalkyl derivative of (II), and reacting theintermediate with phosphorus oxychloride and a suitable heteroaryl suchas pyridine, pyrrole, oxazolyl, imidazolyl, and the like. The reactionmay be carried out at temperatures less than room temperature. Thetemperature is generally from about −20° C. to about 20° C., preferablyabout −10° C. to about 10° C. By “about” is meant that the temperaturerange is preferably within 10 degrees Celsius of the indicatedtemperature, more preferably within 5 degrees Celsius of the indicatedtemperature and, most preferably, within 2 degrees Celsius of theindicated temperature. Thus, for example, by “about 0° C.” is meant 0°C.±10° C., preferably 0° C.±5° C. and most preferably, 0° C.±2° C.

Compounds of Formula (II) can be prepared by methods well known in theart. For example, compound (II) where R³-R⁶, R⁷, and R⁹ are hydrogen andR⁸ and R¹⁰ are methyl can be prepared by following the proceduredescribed in U.S. Pat. No. 5,792,783, at column 22, lines 60-67, thedisclosure of which is incorporated herein by reference. Other compoundsof Formula (II) can be prepared as described in U.S. Pat. No. 5,792,783,PCT Application Publication No. WO 99/61422, and U.S. patent applicationSer. No. 09/783,264, filed on Feb. 15, 2001, and titled “PYRROLESUBSTITUTED 2-INDOLINONE AS PROTEIN KINASE INHIBITORS”, the disclosuresof which are hereby incorporated by reference.

The preparation of compounds of Formula (I) may further include the stepof removing a protecting group. “Protecting group” refers to a groupused to render a reactive moiety inert until removal of the group.Reactive moieties are well known to the skilled artisan; preferredreactive moieties include reactive nitrogen, oxygen, sulfur, carboxyland carbonyl groups. Exemplary nitrogen protecting groups include, butare not limited to, benzyl, benzyloxycarbonyl, tert-butoxycarbonyl,silyl groups (e.g., tert-butyldimethylsilyl),9-fluorenylmethoxycarbonyl, 9-phenyl-9-fluorenyl and arylsulfonyl groups(e.g., toluenesulfonyl). Exemplary oxygen protecting groups include, butare not limited to, allyloxycarbonyl, benzoyl, benzyl, tert-butyl, silylgroups (e.g., tert-butyldimethylsilyl), 2-ethoxyethyl, p-methoxybenzyl,methoxymethyl, pivaloyl, tetrahydropyran-2-yl and trityl. Exemplarycarboxyl protecting groups include, without limitation, methyl, allyl,benzyl, silyl groups (e.g., tert-butyldimethylsilyl) and p-nitrobenzyl.Exemplary carbonyl protecting groups include, but are not limited to,acetyl groups (e.g., O,O-acetals).

Protecting groups may be removed using methods known in the literature.For example, for the removal of nitrogen protecting groups see Greene etal. (1991) Protecting Groups in Organic Synthesis, 2^(nd) ed., JohnWiley & Sons, New York, pp. 309-405 and Kocienski (1994) ProtectingGroups, Thieme, New York, pp. 185-243. Methods for the removal ofparticular protecting groups are exemplified herein.

Utility

The PKs whose catalytic activity is modulated by the compounds of thisinvention include protein tyrosine kinases of which there are two types,receptor tyrosine kinases (RTKs) and cellular tyrosine kinases (CTKs),and serine-threonine kinases (STKs). RTK mediated signal transduction,is initiated by extracellular interaction with a specific growth factor(ligand), followed by receptor dimerization, transient stimulation ofthe intrinsic protein tyrosine kinase activity and phosphorylation.Binding sites are thereby created for intracellular signal transductionmolecules and lead to the formation of complexes with a spectrum ofcytoplasmic signaling molecules that facilitate the appropriate cellularresponse (e.g., cell division, metabolic effects on the extracellularmicroenvironment, etc.). See, Schlessinger and Ullrich, 1992, Neuron9:303-391.

It has been shown that tyrosine phosphorylation sites on growth factorreceptors function as high-affinity binding sites for SH2 (src homology)domains of signaling molecules. Fantl et al., 1992, Cell 69:413-423,Songyang et al., 1994, Mol. Cell. Biol. 14:2777-2785), Songyang et al.,1993, Cell 72:767-778, and Koch et al., 1991, Science 252:668-678.Several intracellular substrate proteins that associate with RTKs havebeen identified. They may be divided into two principal groups: (1)substrates that have a catalytic domain, and (2) substrates which lacksuch domain but which serve as adapters and associate with catalyticallyactive molecules. Songyang et al., 1993, Cell 72:767-778. Thespecificity of the interaction substrates is determined by the aminoacid residues immediately surrounding the phosphorylated tyrosineresidue. Differences in the binding affinities between SH2 domains andthe amino acid sequences surrounding the phosphotyrosine residues onparticular receptors are consistent with the observed differences intheir substrate phosphorylation profiles. Songyang et al., 1993, Cell72:767-778. These observations suggest that the function of each RTK isdetermined not only by its pattern of expression and ligand availabilitybut also by the array of downstream signal transduction pathways thatare activated by a particular receptor. Thus, phosphorylation providesan important regulatory step which determines the selectivity ofsignaling pathways recruited by specific growth factor receptors, aswell as differentiation factor receptors.

STKs, being primarily cytosolic, affect the internal biochemistry of thecell, often as a down-line response to a PTK event. STKs have beenimplicated in the signaling process which initiates DNA synthesis andsubsequent mitosis leading to cell proliferation.

Thus, PK signal transduction results in, among other responses, cellproliferation, differentiation, growth and metabolism. Abnormal cellproliferation may result in a wide array of disorders and diseases,including the development of neoplasia such as carcinoma, sarcoma,glioblastoma and hemangioma, disorders such as leukemia, psoriasis,arteriosclerosis, arthritis and diabetic retinopathy and other disordersrelated to uncontrolled angiogenesis and/or vasculogenesis.

In another aspect, the protein kinase, the catalytic activity of whichis modulated by contact with a compound of this invention, is a proteintyrosine kinase, more particularly, a receptor protein tyrosine kinase.Among the receptor protein tyrosine kinases whose catalytic activity canbe modulated with a compound of this invention, or salt thereof, are,without limitation, EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFRα,PDGFRβ, CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R,FGFR-2R, FGFR-3R and FGFR-4R.

The protein tyrosine kinase whose catalytic activity is modulated bycontact with a compound of this invention, or a salt thereof, can alsobe a non-receptor or cellular protein tyrosine kinase (CTK). Thus, thecatalytic activity of CTKs such as, without limitation, Src, Frk, Btk,Csk, Abl, ZAP70, Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck,Fgr and Yrk, may be modulated by contact with a compound or salt of thisinvention.

Still another group of PKs which may have their catalytic activitymodulated by contact with a compound of this invention are theserine-threonine protein kinases such as, without limitation, CDK2 andRaf.

In another aspect, this invention relates to a method for treating orpreventing a PK related disorder by administering a therapeuticallyeffective amount of a compound of this invention, or a salt thereof, toan organism.

It is also an aspect of this invention that a pharmaceutical compositioncontaining a compound of this invention, or a salt thereof, isadministered to an organism for the purpose of preventing or treating aPK related disorder.

This invention is therefore directed to compounds that modulate PKsignal transduction by affecting the enzymatic activity of RTKs, CTKsand/or STKs, thereby interfering with the signals transduced by suchproteins. More particularly, the present invention is directed tocompounds which modulate RTK, CTK and/or STK mediated signaltransduction pathways as a therapeutic approach to cure many kinds ofsolid tumors, including but not limited to carcinomas, sarcomasincluding Kaposi's sarcoma, erythroblastoma, glioblastoma, meningioma,astrocytoma, melanoma and myoblastoma. Treatment or prevention ofnon-solid tumor cancers such as leukemia are also contemplated by thisinvention. Indications may include, but are not limited to braincancers, bladder cancers, ovarian cancers, gastric cancers, pancreascancers, colon cancers, blood cancers, lung cancers and bone cancers.

Further examples, without limitation, of the types of disorders relatedto inappropriate PK activity that the compounds described herein may beuseful in preventing, treating and studying, are cell proliferativedisorders, fibrotic disorders, metabolic disorders and infectiousdiseases.

Cell proliferative disorders, which may be prevented, treated or furtherstudied by the present invention include cancer, blood vesselproliferative disorders and mesangial cell proliferative disorders.

Blood vessel proliferative disorders refer to disorders related toabnormal vasculogenesis (blood vessel formation) and angiogenesis(spreading of blood vessels). While vasculogenesis and angiogenesis playimportant roles in a variety of normal physiological processes such asembryonic development, corpus luteum formation, wound healing and organregeneration, they also play a pivotal role in cancer development wherethey result in the formation of new capillaries needed to keep a tumoralive. Other examples of blood vessel proliferation disorders includearthritis, where new capillary blood vessels invade the joint anddestroy cartilage, and ocular diseases, like diabetic retinopathy, wherenew capillaries in the retina invade the vitreous, bleed and causeblindness.

Two structurally related RTKs have been identified to bind VEGF withhigh affinity: the fins-like tyrosine 1 (fit-1) receptor (Shibuya etal., 1990, Oncogene,5:519-524; De Vries et al., 1992, Science,255:989-991) and the KDR/FLK-1 receptor, also known as VEGF-R2. Vascularendothelial growth factor (VEGF) has been reported to be an endothelialcell specific mitogen with in vitro endothelial cell growth promotingactivity. Ferrara & Henzel, 1989, Biochem. Biophys. Res. Comm.,161:851-858; Vaisman et al., 1990, J. Biol. Chem., 265:19461-19566.Information set forth in U.S. application Ser. Nos. 08/193,829,08/038,596 and 07/975,750, strongly suggest that VEGF is not onlyresponsible for endothelial cell proliferation, but also is the primeregulator of normal and pathological angiogenesis. See generally,Klagsburn & Soker, 1993, Current Biology, 3(10)699-702; Houck, et al.,1992, J. Biol. Chem., 267:26031-26037.

Normal vasculogenesis and angiogenesis play important roles in a varietyof physiological processes such as embryonic development, wound healing,organ regeneration and female reproductive processes such as follicledevelopment in the corpus luteum during ovulation and placental growthafter pregnancy. Folkman & Shing, 1992, J. Biological Chem.,267(16):10931-34. Uncontrolled vasculogenesis and/or angiogenesis hasbeen associated with diseases such as diabetes as well as with malignantsolid tumors that rely on vascularization for growth. Klagsburn & Soker,1993, Current Biology, 3(10):699-702; Folkham, 1991, J. Natl. CancerInst., 82:4-6; Weidner, et al., 1991, New Engl. J. Med., 324:1-5.

As presently understood, the role of VEGF in endothelial cellproliferation and migration during angiogenesis and vasculogenesisindicates an important role for the KDR/FLK-1 receptor in theseprocesses. Diseases such as diabetes mellitus (Folkman, 198, in XIthCongress of Thrombosis and Haemostasis (Verstraeta, et al., eds.), pp.583-596, Leuven University Press, Leuven) and arthritis, as well asmalignant tumor growth may result from uncontrolled angiogenesis. Seee.g., Folkman, 1971, N. Engl. J. Med., 285:1182-1186. The receptors towhich VEGF specifically binds are an important and powerful therapeutictarget for the regulation and modulation of vasculogenesis and/orangiogenesis and a variety of severe diseases which involve abnormalcellular growth caused by such processes. Plowman, et al., 1994, DN&P,7(6):334-339. More particularly, the KDR/FLK-1 receptor's highlyspecific role in neovascularization make it a choice target fortherapeutic approaches to the treatment of cancer and other diseaseswhich involve the uncontrolled formation of blood vessels.

Thus, one aspect of the present invention relates to compounds capableof regulating and/or modulating tyrosine kinase signal transductionincluding KDR/FLK-1 receptor signal transduction in order to inhibit orpromote angiogenesis and/or vasculogenesis, that is, compounds thatinhibit, prevent, or interfere with the signal transduced by KDR/FLK-1when activated by ligands such as VEGF. Although it is believed that thecompounds of the present invention act on a receptor or other componentalong the tyrosine kinase signal transduction pathway, they may also actdirectly on the tumor cells that result from uncontrolled angiogenesis.

Although the nomenclature of the human and murine counterparts of thegeneric “flk-1” receptor differ, they are, in many respects,interchangeable. The murine receptor, Flk-1, and its human counterpart,KDR, share a sequence homology of 93.4% within the intracellular domain.Likewise, murine FLK-I binds human VEGF with the same affinity as mouseVEGF, and accordingly, is activated by the ligand derived from eitherspecies. Millauer et al., 1993, Cell, 72:835-846; Quinn et al., 1993,Proc. Natl. Acad. Sci. USA, 90:7533-7537. FLK-1 also associates with andsubsequently tyrosine phosphorylates human RTK substrates (e.g., PLC-γor p85) when co-expressed in 293 cells (human embryonal kidneyfibroblasts).

Models which rely upon the FLK-1 receptor therefore are directlyapplicable to understanding the KDR receptor. For example, use of themurine FLK-1 receptor in methods which identify compounds that regulatethe murine signal transduction pathway are directly applicable to theidentification of compounds which may be used to regulate the humansignal transduction pathway, that is, which regulate activity related tothe KDR receptor. Thus, chemical compounds identified as inhibitors ofKDR/FLK-1 in vitro, can be confirmed in suitable in vivo models. Both invivo mouse and rat animal models have been demonstrated to be ofexcellent value for the examination of the clinical potential of agentsacting on the KDR/FLK-1 induced signal transduction pathway.

Thus, in one aspect, this invention is directed to compounds thatregulate, modulate and/or inhibit vasculogenesis and/or angiogenesis byaffecting the enzymatic activity of the KDR/FLK-1 receptor andinterfering with the signal transduced by KDR/FLK-1. In another aspect,the present invention is directed to compounds which regulate, modulateand/or inhibit the KDR/FLK-1 mediated signal transduction pathway as atherapeutic approach to the treatment of many kinds of solid tumorsincluding, but not limited to, glioblastoma, melanoma and Kaposi'ssarcoma, and ovarian, lung, mammary, prostate, pancreatic, colon andepidermoid carcinoma. In addition, data suggest the administration ofcompounds which inhibit the KDR/Flk-1 mediated signal transductionpathway may also be used in the treatment of hemangioma, restenois anddiabetic retinopathy.

A further aspect of this invention relates to the inhibition ofvasculogenesis and angiogenesis by other receptor-mediated pathways,including the pathway comprising the flt-1 receptor.

Receptor tyrosine kinase mediated signal transduction is initiated byextracellular interaction with a specific growth factor (ligand),followed by receptor dimerization, transient stimulation of theintrinsic protein tyrosine kinase activity and autophosphorylation.Binding sites are thereby created for intracellular signal transductionmolecules which leads to the formation of complexes with a spectrum ofcytoplasmic signaling molecules that facilitate the appropriate cellularresponse, e.g., cell division and metabolic effects to the extracellularmicroenvironment. See, Schlessinger and Ullrich, 1992, Neuron, 9:1-20.

The close homology of the intracellular regions of KDR/FLK-1 with thatof the PDGF-β receptor (50.3% homology) and/or the related flt-1receptor indicates the induction of overlapping signal transductionpathways. For example, for the PDGF-β receptor, members of the srcfamily (Twamley et al., 1993, Proc. Natl. Acad. Sci. USA, 90:7696-7700),phosphatidylinositol-3′-kinase (Hu et al., 1992, Mol. Cell. Biol.,12:981-990), phospholipase cγ (Kashishian & Cooper, 1993, Mol. Cell.Biol., 4:49-51), ras-GTPase-activating protein, (Kashishian et al.,1992, EMBO J., 11:1373-1382), PTP-ID/syp (Kazlauskas et al., 1993, Proc.Natl. Acad. Sci. USA, 10 90:6939-6943), Grb2 (Arvidsson et al., 1994,Mol. Cell. Biol., 14:6715-6726), and the adapter molecules Shc and Nck(Nishimura et al., 1993, Mol. Cell. Biol., 13:6889-6896), have beenshown to bind to regions involving different autophosphorylation sites.See generally, Claesson-Welsh, 1994, Prog. Growth Factor Res., 5:37-54.Thus, it is likely that signal transduction pathways activated byKDR/FLK-1 include the ras pathway (Rozakis et al., 1992, Nature,360:689-692), the PI-3′-kinase, the src-mediated and the plcγ-mediatedpathways. Each of these pathways may play a critical role in theangiogenic and/or vasculogenic effect of KDR/FLK-1 in endothelial cells.Consequently, a still further aspect of this invention relates to theuse of the organic compounds described herein to modulate angiogenesisand vasculogenesis as such processes are controlled by these pathways.

Conversely, disorders related to the shrinkage, contraction or closingof blood vessels, such as restenosis, are also implicated and may betreated or prevented by the methods of this invention.

Fibrotic disorders refer to the abnormal formation of extracellularmatrices. Examples of fibrotic disorders include hepatic cirrhosis andmesangial cell proliferative disorders. Hepatic cirrhosis ischaracterized by the increase in extracellular matrix constituentsresulting in the formation of a hepatic scar. An increased extracellularmatrix resulting in a hepatic scar can also be caused by a viralinfection such as hepatitis. Lipocytes appear to play a major role inhepatic cirrhosis. Other fibrotic disorders implicated includeatherosclerosis.

Mesangial cell proliferative disorders refer to disorders brought aboutby abnormal proliferation of mesangial cells. Mesangial proliferativedisorders include various human renal diseases such asglomerulonephritis, diabetic nephropathy and malignant nephrosclerosisas well as such disorders as thrombotic microangiopathy syndromes,transplant rejection, and glomerulopathies. The RTK PDGFR has beenimplicated in the maintenance of mesangial cell proliferation. Floege etal., 1993, Kidney International 43:47S-54S.

Many cancers are cell proliferative disorders and, as noted previously,PKs have been associated with cell proliferative disorders. Thus, it isnot surprising that PKs such as, for example, members of the RTK familyhave been associated with the development of cancer. Some of thesereceptors, like EGFR (Tuzi et al., 1991, Br. J. Cancer 63:227-233, Torpet al, 1992, APMIS 100:713-719) HER2/neu (Slamon et al., 1989, Science244:707-712) and PDGF-R (Kumabe et al., 1992, Oncogene, 7:627-633) areover-expressed in many tumors and/or persistently activated by autocrineloops. In fact, in the most common and severe cancers these receptorover-expressions (Akbasak and Suner-Akbasak et al., 1992, J. Neurol.Sci., 111:119-133, Dickson et al., 1992, Cancer Treatment Res.61:249-273, Korc et al., 1992, J. Clin. Invest. 90:1352-1360) andautocrine loops (Lee and Donoghue, 1992, J. Cell. Biol., 118:1057-1070,Korc et al., supra, Akbasak and Suner-Akbasak et al., supra) have beendemonstrated. For example, EGFR has been associated with squamous cellcarcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancerand bladder cancer. HER2 has been associated with breast, ovarian,gastric, lung, pancreas and bladder cancer. PDGFR has been associatedwith glioblastoma and melanoma as well as lung, ovarian and prostatecancer. The RTK c-met has also been associated with malignant tumorformation. For example, c-met has been associated with, among othercancers, colorectal, thyroid, pancreatic, gastric and hepatocellularcarcinomas and lymphomas. Additionally c-met has been linked toleukemia. Over-expression of the c-met gene has also been detected inpatients with Hodgkins disease and Burkitts disease.

IGF-IR, in addition to being implicated in nutritional support and intype-II diabetes, has also been associated with several types ofcancers. For example, IGF-I has been implicated as an autocrine growthstimulator for several tumor types, e.g. human breast cancer carcinomacells (Arteaga et al., 1989, J. Clin. Invest. 84:1418-1423) and smalllung tumor cells (Macauley et al., 1990, Cancer Res., 50:2511-2517). Inaddition, IGF-I, while integrally involved in the normal growth anddifferentiation of the nervous system, also appears to be an autocrinestimulator of human gliomas. Sandberg-Nordqvist et al., 1993, CancerRes. 53:2475-2478. The importance of IGF-IR and its ligands in cellproliferation is further supported by the fact that many cell types inculture (fibroblasts, epithelial cells, smooth muscle cells,T-lymphocytes, myeloid cells, chondrocytes and osteoblasts (the stemcells of the bone marrow)) are stimulated to grow by IGF-I. Goldring andGoldring, 1991, Eukaryotic Gene Expression,1:301-326. In a series ofrecent publications, Baserga suggests that IGF-IR plays a central rolein the mechanism of transformation and, as such, could be a preferredtarget for therapeutic interventions for a broad spectrum of humanmalignancies. Baserga, 1995, Cancer Res., 55:249-252, Baserga, 1994,Cell 79:927-930, Coppola et aL, 1994, Mol. Cell. Biol., 14:4588-4595.

STKs have been implicated in many types of cancer including, notably,breast cancer (Cance, et al., Int. J. Cancer, 54:571-77 (1993)).

The association between abnormal PK activity and disease is notrestricted to cancer. For example, RTKs have been associated withdiseases such as psoriasis, diabetes mellitus, endometriosis,angiogenesis, atheromatous plaque development, Alzheimer's disease, vonHippel-Lindau disease, epidermal hyperproliferation, neurodegenerativediseases, age-related macular degeneration and hemangiomas. For example,EGFR has been indicated in corneal and dermal wound healing. Defects inInsulin-R and IGF-TR are indicated in type-II diabetes mellitus. A morecomplete correlation between specific RTKs and their therapeuticindications is set forth in Plowman et al., 1994, DN&P 7:334-339.

As noted previously, not only RTKs but CTKs including, but not limitedto, src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr and yrk (reviewedby Bolen et al., 1992, FASEB J., 6:3403-3409) are involved in theproliferative and metabolic signal transduction pathway and thus couldbe expected, and have been shown, to be involved in many PTK-mediateddisorders to which the present invention is directed. For example,mutated src (v-src) has been shown to be an oncoprotein (pp60^(v-src))in chicken. Moreover, its cellular homolog, the proto-oncogene pp₆₀^(c-src) transmits oncogenic signals of many receptors. Over-expressionof EGFR or HER2/neu in tumors leads to the constitutive activation ofpp60^(c-src), which is characteristic of malignant cells but absent innormal cells. On the other hand, mice deficient in the expression ofc-src exhibit an osteopetrotic phenotype, indicating a key participationof c-src in osteoclast function and a possible involvement in relateddisorders.

Similarly, Zap70 has been implicated in T-cell signaling which mayrelate to autoimmune disorders.

STKs have been associated with inflammation, autoimmune disease,immunoresponses, and hyperproliferation disorders such as restenosis,fibrosis, psoriasis, osteoarthritis and rheumatoid arthritis.

PKs have also been implicated in embryo implantation. Thus, thecompounds of this invention may provide an effective method ofpreventing such embryo implantation and thereby be useful as birthcontrol agents.

In yet another aspect, the compounds of the instant invention can alsobe used as anti-infective agents. For example, indolinone compounds areknown to exhibit antibacterial and antifungal activities. See, e.g.,Singh and Jha (1989) “Indolinone derivatives as potential antimicrobialagents,” Zentralbl. Mikrobiol. 144(2):105-109. In addition, indolinonecompounds have been reported to exhibit significant antiviral activity.See, e.g., Maass et al. (1993) “Viral resistance to thethiazolo-iso-indolinones, a new class of nonnucleoside inhibitors ofhuman immunodeficiency virus type I reverse transcriptase,” Antimicrob.Agents Chemother. 37(12):2612-2617.

Finally, both RTKs and CTKs are currently suspected as being involved inhyperimmune disorders.

A method for identifying a chemical compound that modulates thecatalytic activity of one or more of the above discussed protein kinasesis another aspect of this invention. The method involves contactingcells expressing the desired protein kinase with a compound of thisinvention (or its salt) and monitoring the cells for any effect that thecompound has on them. The effect may be any observable, either to thenaked eye or through the use of instrumentation, change or absence ofchange in a cell phenotype. The change or absence of change in the cellphenotype monitored may be, for example, without limitation, a change orabsence of change in the catalytic activity of the protein kinase in thecells or a change or absence of change in the interaction of the proteinkinase with a natural binding partner.

Pharmaceutical Compositions and Administration

A compound of the present invention or a physiologically acceptable saltthereof, can be administered as such to a human patient or can beadministered in pharmaceutical compositions in which the foregoingmaterials are mixed with suitable carriers or excipient(s). Techniquesfor formulation and administration of drugs may be found in “Remington'sPharmacological Sciences,” Mack Publishing Co., Easton, Pa., latestedition.

Routes of Administration

As used herein, “administer” or “administration” refers to the deliveryof a compound or salt of the present invention or of a pharmaceuticalcomposition containing a compound or salt of this invention to anorganism for the purpose of prevention or treatment of a PK-relateddisorder.

Suitable routes of administration may include, without limitation, oral,rectal, transmucosal or intestinal administration or intramuscular,subcutaneous, intramedullary, intrathecal, direct intraventricular,intravenous, intravitreal, intraperitoneal, intranasal, or intraocularinjections. The preferred routes of administration are oral andparenteral.

Alternatively, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a solid tumor, often in a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with tumor-specific antibody.The liposomes will be targeted to and taken up selectively by the tumor.

Composition/Formulation

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, lyophilizing processes or spray drying.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the compounds of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchbuffers with or without a low concentration of surfactant or cosolvent,or physiological saline buffer. For transmucosal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated by combiningthe active compounds with pharmaceutically acceptable carriers wellknown in the art. Such carriers enable the compounds of the invention tobe formulated as tablets, pills, lozenges, dragees, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient. Pharmaceutical preparations for oral use can be made using asolid excipient, optionally grinding the resulting mixture, andprocessing the mixture of granules, after adding other suitableauxiliaries if desired, to obtain tablets or dragee cores. Usefulexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol, cellulose preparations such as,for example, maize starch, wheat starch, rice starch and potato starchand other materials such as gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinyl-pyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginicacid. A salt such as sodium alginate may also be used.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with a fillersuch as lactose, a binder such as starch, and/or a lubricant such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, liquid polyethyleneglycols, cremophor, capmul, medium or long chain mono- di- ortriglycerides. Stabilizers may be added in these formulations, also.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray using a pressurized pack or a nebulizer and a suitable propellant,e.g., without limitation, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetra-fluoroethane or carbon dioxide. Inthe case of a pressurized aerosol, the dosage unit may be controlled byproviding a valve to deliver a metered amount. Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

The compounds may also be formulated for parenteral administration, e.g.by bolus injection or continuous infusion. Formulations for injectionmay be presented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulating materials such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of a water soluble form, such as, without limitation,a salt, of the active compound. Additionally, suspensions of the activecompounds may be prepared in a lipophilic vehicle. Suitable lipophilicvehicles include fatty oils such as sesame oil, synthetic fatty acidesters such as ethyl oleate and triglycerides, or materials such asliposomes. Aqueous injection suspensions may contain substances whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers and/or agents that increase the solubilityof the compounds to allow for the preparation of highly concentratedsolutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterwith or without additional sufactants or cosolvents such as polysorbate80, Cremophor, cyclodextrin sulfobutylethyl, propylene glycol, orpolyethylene glycol e.g., PEG-300 or PEG 400, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, using, e.g., conventional suppositorybases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as depot preparations. Such long acting formulationsmay be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. A compound of thisinvention may be formulated for this route of administration withsuitable polymeric or hydrophobic materials (for instance, in anemulsion with a pharmacologically acceptable oil), with ion exchangeresins, or as a sparingly soluble derivative such as, withoutlimitation, a sparingly soluble salt.

A non-limiting example of a pharmaceutical carrier for the hydrophobiccompounds of the invention is a cosolvent system comprising benzylalcohol, a nonpolar surfactant, a water-miscible organic polymer and anaqueous phase such as the VPD co-solvent system. VPD is a solution of 3%w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™,and 65% w/v polyethylene glycol 300, made up to volume in absoluteethanol. The VPD co-solvent system (VPD:D5W) consists of VPD diluted 1:1with a 5% dextrose in water solution. This co-solvent system dissolveshydrophobic compounds well, and itself produces low toxicity uponsystemic administration. Naturally, the proportions of such a co-solventsystem may be varied considerably without destroying its solubility andtoxicity characteristics. Furthermore, the identity of the co-solventcomponents may be varied: for example, other low-toxicity nonpolarsurfactants may be used instead of Polysorbate 80™, the fraction size ofpolyethylene glycol may be varied, other biocompatible polymers mayreplace polyethylene glycol, e.g., polyvinyl pyrrolidone, and othersugars or polysaceharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are well knownexamples of delivery vehicles or carriers for hydrophobic drugs. Inaddition, certain organic solvents such as dimethylsulfoxide also may beemployed, although often at the cost of greater toxicity.

Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various sustained-release materialshave been established and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

The pharmaceutical compositions herein also may comprise suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include, but are not limited to, calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols.

Many of the PK modulating compounds of the invention may be provided asphysiologically acceptable salts wherein the claimed compound may formthe negatively or the positively charged species. Examples of salts inwhich the compound forms the positively charged moiety include, withoutlimitation, quaternary ammonium (defined elsewhere herein), salts suchas the hydrochloride, sulfate, citrate, mesylate, lactate, tartrate,maleate, succinate wherein the nitrogen atom of the quaternary ammoniumgroup is a nitrogen of the selected compound of this invention which hasreacted with the appropriate acid. Salts in which a compound of thisinvention forms the negatively charged species include, withoutlimitation, the sodium, potassium, calcium and magnesium salts formed bythe reaction of a carboxylic acid group in the compound with anappropriate base (e.g. sodium hydroxide (NaOH), potassium hydroxide(KOH), Calcium hydroxide (Ca(OH)₂), etc.).

Dosage

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in anamount sufficient to achieve the intended purpose, i.e., the modulationof PK activity or the treatment or prevention of a PK-related disorder.

More specifically, a therapeutically effective amount means an amount ofcompound effective to prevent, alleviate or ameliorate symptoms ofdisease or prolong the survival of the subject being treated.Therapeutically effective amounts of compounds of Formula I may rangefrom approximately 10/m² to 400/m², preferably 50/M² to 300/m², morepreferably 100/m² to 220/m², even more preferably 195/m².

For any compound used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromcell culture assays. Then, the dosage can be formulated for use inanimal models so as to achieve a circulating concentration range thatincludes the IC₅₀ as determined in cell culture (i.e., the concentrationof the test compound which achieves a half-maximal inhibition of the PKactivity). Such information can then be used to more accuratelydetermine useful doses in humans.

Toxicity and therapeutic efficacy of the compounds described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., by determining the IC₅₀, and the LD₅₀ (bothof which are discussed elsewhere herein) for a subject compound. Thedata obtained from these cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage mayvary depending upon the dosage form employed and the route ofadministration utilized. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition. (See e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p.1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active species which are sufficient to maintain thekinase modulating effects. These plasma levels are referred to asminimal effective concentrations (MECs). The MEC will vary for eachcompound but can be estimated from in vitro data, e.g., theconcentration necessary to achieve 50-90% inhibition of a kinase may beascertained using the assays described herein. Preferably, the Dosagesnecessary to achieve the MEC will depend on individual characteristicsand route of administration. HPLC assays or bioassays can be used todetermine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compoundsshould be administered using a regimen that maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%. In cases of local administration or selectiveuptake, the effective local concentration of the drug may not be relatedto plasma concentration and other procedures known in the art may beemployed to determine the correct dosage amount and interval.

The amount of a composition administered will, of course, be dependenton the subject being treated, the severity of the affliction, the mannerof administration, the judgment of the prescribing physician, etc.

Packaging

The compositions may, if desired, be presented in a pack or dispenserdevice, such as an FDA approved kit, which may contain one or more unitdosage forms containing the active ingredient. The pack may for examplecomprise metal or plastic foil, such as a blister pack. The pack ordispenser device may be accompanied by instructions for administration.The pack or dispenser may also be accompanied by a notice associatedwith the container in a form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals, which noticeis reflective of approval by the agency of the form of the compositionsor of human or veterinary administration. Such notice, for example, maybe of the labeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert. Compositionscomprising a compound of the invention formulated in a compatiblepharmaceutical carrier may also be prepared, placed in an appropriatecontainer, and labeled for treatment of an indicated condition. Suitableconditions indicated on the label may include treatment of a tumor,inhibition of angiogenesis, treatment of fibrosis, diabetes, and thelike.

It is also an aspect of this invention that a compound described herein,or its salt, might be combined with other chemotherapeutic agents forthe treatment of the diseases and disorders discussed above. Forinstance, a compound or salt of this invention might be combined withalkylating agents such as fluorouracil (5-FU) alone or in furthercombination with leukovorin; or other alkylating agents such as, withoutlimitation, other pyrimidine analogs such as UFT, capecitabine,gemcitabine and cytarabine, the alkyl sulfonates, e.g., busulfan (usedin the treatment of chronic granulocytic leukemia), improsulfan andpiposulfan; aziridines, e.g., benzodepa, carboquone, meturedepa anduredepa; ethyleneimines and methylmelamines, e.g., altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylolmelamine; and the nitrogenmustards, e.g., chlorambucil (used in the treatment of chroniclymphocytic leukemia, primary macroglobulinemia and non-Hodgkin'slymphoma), cyclophosphamide (used in the treatment of Hodgkin's disease,multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lungcancer, Wilm's tumor and rhabdomyosarcoma), estramustine, ifosfamide,novembrichin, prednimustine and uracil mustard (used in the treatment ofprimary thrombocytosis, non-Hodgkin's lymphoma, Hodgkin's disease andovarian cancer); and triazines, e.g., dacarbazine (used in the treatmentof soft tissue sarcoma).

Likewise a compound or salt of this invention might be expected to havea beneficial effect in combination with other antimetabolitechemotherapeutic agents such as, without limitation, folic acid analogs,e.g. methotrexate (used in the treatment of acute lymphocytic leukemia,choriocarcinoma, mycosis fungiodes breast cancer, head and neck cancerand osteogenic sarcoma) and pteropterin; and the purine analogs such asmercaptopurine and thioguanine which find use in the treatment of acutegranulocytic, acute lymphocytic and chronic granulocytic leukemias.

A compound or salt of this invention might also be expected to proveefficacious in combination with natural product based chemotherapeuticagents such as, without limitation, the vinca alkaloids, e.g.,vinblastin (used in the treatment of breast and testicular cancer),vincristine and vindesine; the epipodophylotoxins, e.g., etoposide andteniposide, both of which are useful in the treatment of testicularcancer and Kaposi's sarcoma; the antibiotic chemotherapeutic agents,e.g., daunorubicin, doxorubicin, epirubicin, mitomycin (used to treatstomach, cervix, colon, breast, bladder and pancreatic cancer),dactinomycin, temozolomide, plicamycin, bleomycin (used in the treatmentof skin, esophagus and genitourinary tract cancer); and the enzymaticchemotherapeutic agents such as L-asparaginase.

In addition to the above, a compound or salt of this invention might beexpected to have a beneficial effect used in combination with theplatinum coordination complexes (cisplatin, etc.); substituted ureassuch as hydroxyurea; methylhydrazine derivatives, e.g., procarbazine;adrenocortical suppressants, e.g., mitotane, aminoglutethimide; andhormone and hormone antagonists such as the adrenocorticosteriods (e.g.,prednisone), progestins (e.g., hydroxyprogesterone caproate); estrogens(e.g., diethylstilbesterol); antiestrogens such as tamoxifen; androgens,e.g., testosterone propionate; and aromatase inhibitors (such asanastrozole).

Finally, the combination of a compound of this invention might beexpected to be particularly effective in combination with Camptosar™,Gleevec™, Herceptin™, Endostatin™, Cox-2 inhibitors, Mitoxantrone™ orPaclitaxel™ for the treatment of solid tumor cancers or leukemias suchas, without limitation, acute myelogenous (non-lymphocytic) leukemia.

EXAMPLES

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representativethereof.

In general HPLC data was obtained with a Zorbax SB C18 column (4.6 mmID×7.5 cm), a Perkin Elmer series 200 pump programmed to run from 10%acetonitrile/water 0.1% TFA (solvent A) to 90% acetonitrile/water(solvent B) with a flow rate of 1.5 mL/min. After 0.1 min on solvent A,a 5 min linear program to solvent B was run, followed by 3 min onsolvent B, before recycling to solvent A (2 min). Detection was with aPerkin Elmer diode array detector recording at 215 and 280 nM). NMRspectra were recorded on a Bruker instrument at 300 MHz.

Synthetic Examples Example 1 Synthesis of(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-[1-(4-methylpiperazinylmethyl]-1,3-dihydro-2H-indol-2-one

N-Methylpiperazine (10 g, 100 mmol) was added to a stirred solution ofaqueous formaldehyde (10 g of 38% solution, 100 mmol) and3-(3,5-dimethyl-1H-pyrrol-2-ylmethylidene)-1,3-dihydro-indol-2-one (U.S.Pat. No. 5,792,783) (2.38 g, 10 mmol) in methanol (100 mL). The solutionheated at 60° C. for 1 h, concentrated to a low volume and theprecipitate was filtered off, washed with methanol, and dried to give2.38 g of the title compound, mp 160-164° C. HPLC Rt 4.72 min. ¹H NMR(CDCl₃) δ2.26 (s, 3H), 2.33 (s, 3H), 2.38 (s, 3H), 2.43 (br s, 4H), 2.70(br s, 4H), 4.59 (s, 2H), 5.96 (d, 1H), 7.02-7.08 (m, 2H), 7.15 (dd,1H), 7.38 (s, 1H), 7.48 (dd, 1H) and 13.0 (br s, 1H). Anal. Calcd forC₂₁H₂₆N₄O: C, 71.97; H, 7.48; N, 15.99. Found: C, 71.75; H, 7.46; N,15.87.

(3Z)-3-[(3,5-Dimethyl-1H-pyrrol-2-yl)-methylidene]-1-[1-(4-methylpiperazinyl)methyl]-1,3-dihydro-2H-indol-2-onewas then converted to a dihydrochloride salt.

Example 2 Synthesis of(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one

Pyrrolidine (450 mg, 6.3 mmol) was added to a stirred solution ofaqueous formaldehyde (500 mg of 38% solution, 6.0 mmol) and3-(3,5-dimethyl-1H-pyrrol-2-ylmethylidene)-1,3-dihydro-indol-2-one (900mg, 3.8 mmol) in methanol (50 mL). After 15 min., the solution wascooled to 0° C. and the precipitate was filtered off, washed with water,and dried to give 1.08 g of the title compound, mp 129-132° C. HPLC Rt4.87 min. ¹H NMR [(CD₃)₂SO] δ1.65 (m, 4H), 2.32 9s, 3H), 2.34 (s, 3H),2.62 (m, 4H), 4.72 (s, 2H) 6.07 (d, 1H), 7.00 (m, 1H), 7.15 (m, 2H),7.61 (s, 1H), 7.76 (d, 2H) and 13.1 (br s, 1H). Anal. Calcd forC₂₀H₂₃N₃O: C, 74.74; H, 7.21; N, 13.07. Found: C, 74.61; H, 7.25; N,13.03.

Following the procedure described above, but substituting pyrrolidinewith 2-hydroxymethylpyrrolidine, 2-methylpyrrolidine,2-methoxymethylpyrrolidine, proline, 3,5-dimethylpiperazine, anbis-(2-methoxyethyl)amine provide:

(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(2-hydroxypyrrolidin-1-ylmethyl)-1,3-dihydro-2H-indol-2-one;

(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(2-methylpyrrolidin-1-ylmethyl)-1,3-dihydro-2H-indol-2-one;

(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(2-methoxymethyl-pyrrolidin-1-ylmethyl)-1,3-dihydro-2H-indol-2-one;

(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(2-carboxypyrrolidin-1-ylmethyl)-1,3-dihydro-2H-indol-2-one;

(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(3,5-dimethylpiperazin-1-ylmethyl)-1,3-dihydro-2H-indol-2-one;and

(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-[bis(2-methoxyethyl-aminomethyl)]-1,3-dihydro-2H-indol-2-onerespectively.

Example 3 Synthesis of1-({(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-2-oxo-1,3-dihydro-1H-indol-1-yl}methyl)pyridiniumChloride

Aqueous formaldehyde (15.0 g of 38% solution, 190 mmol) was added to astirred solution of3(Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1,3-dihydro-2H-indol-2-one(23.8 g, 100 mmol) and triethylamine (15.0 g, 150 mmol) indimethylformamide (200 mL). After 1 h, the solution was diluted withwater and the precipitate was filtered off, washed with water, and driedto give 26.4 g of3(Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(hydroxymethyl)-1,3-dihydro-2H-indol-2-one,mp 196-200° C. HPLC Rt 5.71 min. ¹H NMR (CDCl₃) δ2.34 (s, 6H), 3.14 (t,1H), 5.44 (d, 2H), 5.98 (d, 1H), 7.08 (m, 2H), 7.18 (m, 1H), 7.36 (s,1H), 7.48 (dd, 1H) and 13.0 (br s, 1H). Anal. Calcd for C₁₆H₁₆N₂O₂: C,71.62; H, 6.01; N, 10.44. Found: C, 71.33; H, 6.09; N, 10.43.

Phosphorus oxychloride (3.1 g, 10 mmol) was added at 0° C. to a stirredsolution of(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-1-(hydroxymethyl)-1,3-dihydro-2H-indol-2-one(2.68 g, 10 mmol) in pyridine (20 mL). After 130 min, the solution wasdiluted slowly with water (20 mL) and the precipitate was filtered off,washed with water, and dried to give 3.3 g of the title compound,mp>280° C. HPLC Rt 4.78 min. ¹H NMR [(CD₃OD] δ2.35 (s, 3H), 2.37 (s,3H), 6.07 (d, 1H), 6.71 (s, 2H), 7.12-7.32 (m, 3H), 7.62 Calcd forC₂₁H₂₀CIN₃O: C, 68.94; H, 5.51; Cl, 9.69; N, 11.48. Found: C, 68.63; H,5.53; Cl, 9.53; N, 11.45.

Other compounds of Formula (II) such as5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)-amide can be prepared as follows:

Hydrazine hydrate (55%, 3000 mL) and 5-fluoroisatin (300 g) were heatedto 100° C. An additional 5-fluoro-isatin (500 g) was added in portions(100 g) over 120 minutes with stirring. The mixture was heated to 110°C. and stirred for 4 hours. The mixture was cooled to room temperatureand the solids collected by vacuum filtration to give crude(2-amino-5-fluoro-phenyl)-acetic acid hydrazide (748 g). The hydrazidewas suspended in water (700 mL) and the pH of the mixture adjusted to<pH 3 with 12 N hydrochloric acid. The mixture was stirred for 12 hoursat room temperature. The solids were collected by vacuum filtration andwashed twice with water. The product was dried under vacuum to give5-fluoro-1,3-dihydro-indol-2-one (600 g, 73% yield) as a brown powder.¹H-NMR (dimethylsulfoxide-d₆) δ3.46 (s, 2H, CH₂), 6.75, 6.95, 7.05 (3×m,3H, aromatic), 10.35 (s, 1H, NH). MS m/z 152 [M+1].

3,5-Dimethyl-1H-pyrrole-2,4-dicarboxylic acid 2-tert-butyl ester 4-ethylester (2600 g) and ethanol (7800 mL) were stirred vigorously while 10 Nhydrochloric acid (3650 mL) was slowly added. The temperature increasedfrom 25° C. to 35° C. and gas evolution began. The mixture was warmed to54° C. and stirred with further heating for one hour at which time thetemperature was 67° C. The mixture was cooled to 5° C. and 32 L of iceand water were slowly added with stirring. The solid was collected byvacuum filtration and washed three times with water. The solid was airdried to constant weight to give 2,4-dimethyl-1H-pyrrole-3-carboxylicacid ethyl ester (1418 g, 87% yield) as a pinkish solid. ¹H-NMR(dimethylsulfoxide-d₆) δ2.10, 2.35 (2×s, 2×3H, 2×CH₃), 4.13 (q, 2H,CH₂), 6.37 (s, 1H, CH), 10.85 (s, 1H, NH). MS m/z 167 [M+1].

Dimethylformamide (322 g) and dichloromethane (3700 mL) were cooled inan ice bath to 4° C. and phosphorus oxychloride (684 g) was added withstirring. Solid 2,4-dimethyl-1H-pyrrole-3-carboxylic acid ethyl ester(670 g) was slowly added in aliquots over 15 minutes. The maximumtemperature reached was 18° C. The mixture was heated to reflux for onehour, cooled to 10° C. in an ice bath and 1.6 L of ice water was rapidlyadded with vigorous stirring. The temperature increased to 15° C. 10 NHydrochloric acid (1.6 L) was added with vigorous stirring. Thetemperature increased to 22° C. The mixture was allowed to stand for 30minutes and the layers allowed to separate. The temperature reached amaximum of 40° C. The aqueous layer was adjusted to pH 12-13 with 10 Npotassium hydroxide (3.8 L) at a rate that allowed the temperature toreach and remain at 55° C. during the addition. After the addition wascomplete the mixture was cooled to 10° C. and stirred for 1 hour. Thesolid was collected by vacuum filtration and washed four times withwater to give 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid ethylester (778 g, 100% yield) as a yellow solid. ¹H-NMR (DMSO-d₆) δ1.25 (t,3H, CH₃), 2.44, 2.48 (2×s, 2×3H, 2×CH₃), 4.16 (q, 2H, CH₂), 9.59 (s, 1H,CHO), 12.15 (br s, 1H, NH). MS m/z 195 [M+1].

5-Formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid ethyl ester (806 g),potassium hydroxide (548 g), water (2400 mL) and methanol (300 mL) wererefluxed for two hours with stirring and then cooled to 8° C. Themixture was extracted twice with dichloromethane. The aqueous layer wasadjusted to pH 4 with 1000 mL of 10 N hydrochloric acid keeping thetemperature under 15° C. Water was added to facilitate stirring. Thesolid was collected by vacuum filtration, washed three times with waterand dried under vacuum at 50° C. to give5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic (645 g, 93.5% yield) acidas a yellow solid. NMR (DMSO-d₆) δ2.40, 2.43 (2×s, 2×3H, 2×CH₃), 9.57(s, 1H, CHO), 12.07 (br s, 2H, NH+COOH). MS m/z 168 [M+1].

5-Formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (1204 g) and 6020 mLof dimethylformamide were stirred at room temperature while1-(3-dimethyl-aminopropyl-3-ethylcarbodiimide hydrochloride (2071 g),hydroxybenzotriazole (1460 g), triethylamine (2016 mL) anddiethylethylenediamine (1215 mL) were added. The mixture was stirred for20 hours at room temperature. The mixture was diluted with 3000 mL ofwater, 2000 mL of brine and 3000 mL of saturated sodium bicarbonatesolution and the pH adjusted to greater than 10 with 10 N sodiumhydroxide. The mixture was extracted twice with 5000 mL each time of 10%methanol in dichloromethane and the extracts combined, dried overanhydrous magnesium sulfate and rotary evaporated to dryness. Themixture was with diluted with 1950 mL of toluene and rotary evaporatedagain to dryness. The residue was triturated with 3:1 hexane:diethylether (4000 mL). The solids were collected by vacuum filtration, washedtwice with 400 mL of ethyl acetate and dried under vacuum at 34° C. for21 hours to give 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylamino-ethyl)-amide (819 g, 43% yield) as a light brown solid.¹H-NMR (dimethylsulfoxide-d₆) δ0.96 (t, 6H, 2×CH₃), 2.31, 2.38 (2×s,2×CH₃), 2.51 (m, 6H 3×CH₂), 3.28 (m, 2H, CH₂), 7.34 (m, 1H, amide NH),9.56 (s, 1H, CHO), 11.86 (s, 1H, pyrrole NH). MS m/z 266 [M+1].

5-Formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylaminoethyl)-amide (809 g), 5-fluoro-1,3-dihydro-indol-2-one(438 g), ethanol (8000 mL) and pyrrolidine (13 mL) were heated at 78° C.for 3 hours. The mixture was cooled to room temperature and the solidscollected by vacuum filtration and washed with ethanol. The solids werestirred with ethanol (5900 mL) at 72° C. for 30 minutes. The mixture wascooled to room temperature. The solids were collected by vacuumfiltration, washed with ethanol and dried under vacuum at 54° C. for 130hours to give5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)-amide (1013 g, 88% yield) as an orangesolid. ¹H-NMR (dimethylsulfoxide-d₆) δ0.98 (t, 6H, 2×CH₃), 2.43, 2.44(2×s, 6H, 2×CH₃), 2.50 (m, 6H, 3×CH₂), 3.28 (q, 2H, CH₂), 6.84, 6.92,7.42, 7.71, 7.50 (5×m, 5H, aromatic, vinyl, CONH), 10.88 (s, 1H, CONH),13.68 (s, 1H, pyrrole NH). MS m/z 397 [M−1].

Yet another compound of Formula (II) such as5-(5-fluoro-2-oxo-1,2-dihydro-indol-3-ylidene-methyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (3-diethylamino-2-hydroxy-propyl)-amide can be prepared as follows:

To 2-chloromethyloxirane (95 g, 1.03 mole) was added a mixture of water(3.08 g, 0.17 mole) and diethylamine (106.2 mL, 1.03 mole) at 30° C. Thereaction mixture was then stirred at 28-35° C. for 6 hour and cooled to20-25° C. to give 1-chloro-3-diethylamino-propan-2-ol.

A solution of sodium hydroxide (47.9 g, 1.2 mole) in 78 mL water wasadded 1-chloro-3-diethylamino-propan-2-ol. The resultant was stirred at20-25° C. for 1 hour, diluted with 178 mL of water and extracted withether twice. The combined ether solution was dried with solid potassiumhydroxide and evaporated to give 135 g of crude product which waspurified by fraction distillation to give pure glycidyldiethylamine (98g, 76%) as an oil.

To the ice-cold solution of ammonium hydroxide (25 mL, 159 mmole) of 25%(w/w) was added glycidyldiethylamine dropwise (3.2 g, 24.8 mmol) over 10minutes. The reaction mixture was stirred at 0-5° C. for 1 hour and thenroom temperature for 14 hours. The resulting reaction mixture wasevaporated and distilled (84-90° C. at 500-600 mT) to yield1-amino-3-diethylamino-propan-2-ol (3.3 g, 92%). MS m/z 147 ([M+1]⁻).

To the solution of 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(100 mg, 0.43 mmol), EDC (122.7 mg, 0.64 mmol) and HOBt (86.5 mg, 0.64mmol) in 1.0 mL of DMF was added 1-amino-3-diethylamino-propan-2-ol(93.2 mg, 0.64 mmol). The resulting reaction solution was stirred atroom temperature overnight and evaporated. The residue was suspended in10 mL of water and filtered. The solid was washed with saturated sodiumbicarbonate and water and dried in a high vaccum oven overnight to givecrude product which was purified on column chromatography eluting with6% methanol-dichlormethane containing triethylamine (2 drops/100 mL of6% methanol-dichloromethane) to give5-(5-fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (3-diethylamino-2-hydroxy-propyl)-amide (62 mg, 34%) as a yellowsolid. ¹H NMR (400 MHz, DMSO-d6) δ13.70 (s, 1H, NH−1′), 10.90 (s, 1H,NH−1), 7.76 (dd, J=2.38, 9.33 Hz, 1H, H−4), 7.72 (s, 1H, vinyl-H), 7.60(m, br., 1H, CONHCH₂CH(OH)—CH₂N(C₂H₅)₂−4′), 6.93 (dt, J=2.38, 8.99 Hz,1H, H−5), 6.85 (dd, J=4.55, 8.99 Hz, 1H, H−6), 3.83 (m, br, 1H, OH),3.33 (m, 4H), 2.67 (m, br, 5H), 2.46 (s, 3H, CH₃), 2.44 (s, 3H, CH₃),1.04 (m, br, 6H, CH₃×2). MS m/z (relative intensity, %) 427 ([M+1]⁻,100).

These above compounds of Formula (II) can be converted to a compound ofFormula (I) where R¹ is hydrogen, and R^(3′) and R^(4′) are methyl orcombine to form a pyrrolidine by following the procedure described inExamples 1 and 2 above but substituting3-(3,5-dimethyl-1H-pyrrol-2-ylmethylidene)-1,3-dihydro-indol-2-one with5-(5-fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (3-diethylamino-2-hydroxy-propyl)-amide or5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylamino-ethyl)-amide.

Other compound of Formula (II) can be prepared as described inApplicants' U.S. patent application Ser. No. 60/268,683, filed on Feb.15, 2001, titled “3-(4-AMIDOPYRROL-2-YLMETHYLIDENE)-2-INDOLINONEDERIVATIVES—PROTEIN KINASE INHIBITORS” filed on Feb. 15, 2001 and U.S.patent application Ser. No. 09/783,264, filed on Feb. 15, 2001, andtitled “PYRROLE SUBSTITUTED 2-INDOLINONE AS PROTEIN KINASE INHIBITORS”,the disclosures of which are hereby incorporated by reference.

Biological Evaluation

It will be appreciated that, in any given series of compounds, a rangeof biological activities will be observed. In its presently preferredembodiments, this invention relates to novel1-susbtituted-3-pyrrolidinyl-2-indolinones capable of generating in vivo3-pyrrolidinyl-2-indolinones capable of modulating, regulating and/orinhibiting protein kinase activity. The following assays may be employedto select those compounds demonstrating the optimal degree of thedesired activity.

Assay Procedures

The following in vitro assays may be used to determine the level ofactivity and effect of the different compounds of the present inventionon one or more of the PKs. Similar assays can be designed along the samelines for any PK using techniques well known in the art.

Several of the assays described herein are performed in an ELISA(Enzyme-Linked Immunosorbent Sandwich Assay) format (Voller, et al.,1980, “Enzyme-Linked Immunosorbent Assay,” Manual of ClinicalImmunology, 2d ed., Rose and Friedman, Am. Soc. Of Microbiology,Washington, D.C., pp. 359-371). The general procedure is as follows: acompound is introduced to cells expressing the test kinase, eithernaturally or recombinantly, for a selected period of time after which,if the test kinase is a receptor, a ligand known to activate thereceptor is added. The cells are lysed and the lysate is transferred tothe wells of an ELISA plate previously coated with a specific antibodyrecognizing the substrate of the enzymatic phosphorylation reaction.Non-substrate components of the cell lysate are washed away and theamount of phosphorylation on the substrate is detected with an antibodyspecifically recognizing phosphotyrosine compared with control cellsthat were not contacted with a test compound.

The presently preferred protocols for conducting the ELISA experimentsfor specific PKs is provided below. However, adaptation of theseprotocols for determining the activity of compounds against other RTKs,as well as for CTKs and STKs, is well within the scope of knowledge ofthose skilled in the art. Other assays described herein measure theamount of DNA made in response to activation of a test kinase, which isa general measure of a proliferative response. The general procedure forthis assay is as follows: a compound is introduced to cells expressingthe test kinase, either naturally or recombinantly, for a selectedperiod of time after which, if the test kinase is a receptor, a ligandknown to activate the receptor is added. After incubation at leastovernight, a DNA labeling reagent such as 5-bromodeoxyuridine (BrdU) orH³-thymidine is added. The amount of labeled DNA is detected with eitheran anti-BrdU antibody or by measuring radioactivity and is compared tocontrol cells not contacted with a test compound.

GST-FLK-1 Bioassay

This assay analyzes the tyrosine kinase activity of GST-Flk1 onpoly(glu-tyr) peptides.

Materials and Reagents

1. Corning 96-well ELISA plates (Corning Catalog No. 25805-96).

2. poly(glu-tyr) 4:1, lyophilizate (Sigma Catalog No. P0275), 1 mg/ml insterile PBS.

3. PBS Buffer: for 1 L, mix 0.2 g KH₂PO₄, 1.15 g Na₂HPO₄, 0.2 g KCl and8 g NaCl in approx. 900 ml dH₂O. When all reagents have dissolved,adjust the pH to 7.2 with HCl. Bring total volume to 1 L with dH₂O.

4. PBST Buffer: to 1 L of PBS Buffer, add 1.0 ml Tween-20.

5. TBB—Blocking Buffer: for 1 L, mix 1.21 g TRIS, 8.77 g NaCl, 1 mlTWEEN-20 in approximately 900 ml dH₂O. Adjust pH to 7.2 with HCl. Add 10g BSA, stir to dissolve. Bring total volume to 1 L with dH₂O. Filter toremove particulate matter.

6. 1% BSA in PBS: add 10 g BSA to approx. 990 ml PBS buffer, stir todissolve. Adjust total volume to 1 L with PBS buffer, filter to removeparticulate matter.

7. 50 mM Hepes pH 7.5.

8. GST-Flk1 cd purified from sf9 recombinant baculovirus transformation(SUGEN, Inc.).

9. 4% DMSO in dH₂O.

10. 10 mM ATP in dH₂O.

11. 40 mM MnCl₂

12. Kinase Dilution Buffer (KDB): mix 10 ml Hepes (pH 7.5), 1 ml 5MNaCl, 40 μL 100 mM sodium orthovanadate and 0.4 ml of 5% BSA in dH₂Owith 88.56 ml dH₂O.

13. NUNC 96-well V bottom polypropylene plates, Applied ScientificCatalog # AS-72092

14. EDTA: mix 14.12 g ethylenediaminetetraacetic acid (EDTA) withapprox. 70 ml dH₂O. Add 10 N NaOH until EDTA dissolves. Adjust pH to8.0. Adjust total volume to 100 ml with dH₂O.

15. 1⁰ and 2⁰ Antibody Dilution Buffer: mix 10 ml of 5% BSA in PBSbuffer with 89.5 ml TBST.

16. Anti-phosphotyrosine rabbit polyclonal antisera (SUGEN, Inc.)

17. Goat anti-rabbit HRP conjugate.

18. ABST solution: To approx. 900 ml dH₂O add 19.21 g citric acid and35.49 g Na₂HPO₄. Adjust pH to 4.0 with phosphoric acid. Add2,2′-Azinobis(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS, Sigma, Cat.No. A-1888, hold for approx. ½ hour, filter.

19. 30% Hydrogen Peroxide.

20. ABST/H₂O₂: add 3 μl of H₂O₂ to 15 ml of ABST solution.

21. 0.2 M HCl.

Procedure

1. Coat Corning 96-well ELISA plates with 2 μg of polyEY in 100 μlPBS/well, hold at room temperature for 2 hours or at 4 C. overnight.Cover plates to prevent evaporation.

2. Remove unbound liquid from wells by inverting plate. Wash once withTBST. Pat the plate on a paper towel to remove excess liquid.

3. Add 100 μl of 1% BSA in PBS to each well. Incubate, with shaking, for1 hr. at room temperature.

4. Repeat step 2.

5. Soak wells with 50 mM HEPES (pH7.5, 150 μl/well).

6. Dilute test compound with dH₂O/4% DMSO to 4 times the desired finalassay concentration in 96-well polypropylene plates.

7. Add 25 μl diluted test compound to each well of ELISA plate. Incontrol wells, place 25 μl of dH₂O/4% DMSO.

8. Dilute GST-Flk1 0.005 μg (5 ng)/well in KDB.

9. Add 50 μl of diluted enzyme to each well.

10. Add 25 μl 0.5 M EDTA to negative control wells.

11. Add 25 μl of 40 mM MnCl₂ with 4× ATP (2 μM) to all wells (100 μlfinal volume, 0.5 μM ATP final concentration in each well).

12. Incubate, with shaking, for 15 minutes at room temperature.

13. Stop reaction by adding 25 μl of 500 mM EDTA to each well.

14. Wash 3× with TBST and pat plate on paper towel to remove excessliquid.

15. Add 100 μl per well anti-phosphotyrosine antisera, 1:10,000 dilutionin antibody dilution buffer. Incubate, with shaking, for 90 min. at roomtemperature.

16. Wash as in step 14.

17. Add 100 μl/well of goat anti-rabbit HRP conjugate (1:6,000 inantibody dilution buffer). Incubate, with shaking, for 90 minutes areroom temperature.

18. Wash as in Step 14.

19. Add 100 μl room temperature ABST/H₂O₂ solution to each well.

20. Incubate, with shaking for 15 to 30 minutes at room temperature.

21. If necessary, stop reaction by adding 100 μl of 0.2 M HCl to eachwell.

22. Read results on Dynatech MR7000 ELISA reader with test filter at 410nM and reference filter at 630 nM.

PYK2 Bioassay

This assay is used to measure the in vitro kinase activity of HAepitope-tagged full length pyk2 (FL.pyk2-HA) in an ELISA assay.

Materials and Reagents

1. Corning 96-well ELISA plates.

2. 12CA5 monoclonal anti-HA antibody (SUGEN, Inc.)

3. PBS (Dulbecco's Phosphate-Buffered Saline (Gibco Catalog #450-1300EB)

4. TBST Buffer: for 1 L, mix 8.766 g NaCl, 6.057 g TRIS and 1 ml of 0.1%Triton X-100 in approx. 900 ml dH₂O. Adjust pH to 7.2, bring volume to 1L.

5. Blocking Buffer: for 1 L, mix 100 g 10% BSA, 12.1 g 100 mM TRIS,58.44 g 1M NaCl and 10 mL of 1% TWEEN-20.

6. FL.pyk2-HA from sf9 cell lysates (SUGEN, Inc.).

7. 4% DMSO in MilliQue H₂O.

8. 10 mM ATP in dH₂O.

9. 1M MnCl₂.

10. 1M MgCl₂.

11. 1M Dithiothreitol (DTT).

12. 10×Kinase buffer phosphorylation: mix 5.0 ml 1M Hepes (pH 7.5), 0.2ml 1M MnCl₂, 1.0 ml 1 M MgCl₂, 1.0 ml 10% Triton X-100 in 2.8 ml dH₂O.Just prior to use, add 0.1 ml 1M DTT.

13. NUNC 96-well V bottom polypropylene plates.

14. 500 mM EDTA in dH₂O.

15. Antibody dilution buffer: for 100 mL, 1 mL 5% BSA/PBS and 1 mL 10%Tween-20 in 88 mL TBS.

16. HRP-conjugated anti-Ptyr (PY99, Santa Cruz Biotech Cat. No.SC-7020).

17. ABTS, Moss, Cat. No. ABST-2000.

18. 10% SDS.

Procedure

1. Coat Corning 96 well ELISA plates with 0.5 μg per well 12CA5 anti-HAantibody in 100 μl PBS. Store overnight at 4° C.

2. Remove unbound HA antibody from wells by inverting plate. Wash platewith dH₂O. Pat the plate on a paper towel to remove excess liquid.

3. Add 150 μl Blocking Buffer to each well. Incubate, with shaking, for30 min at room temperature.

4. Wash plate 4× with TBS-T.

5. Dilute lysate in PBS (1.5 μg lysate/100 μl PBS).

6. Add 100 μl of diluted lysate to each well. Shake at room temperaturefor 1 hr.

7. Wash as in step 4.

8. Add 50 μl of 2× kinase Buffer to ELISA plate containing capturedpyk2-HA.

9. Add 25 μL of 400 μM test compound in 4% DMSO to each well. Forcontrol wells use 4% DMSO alone.

10. Add 25 μL of 0.5 M EDTA to negative control wells.

11. Add 25 μl of 20 μM ATP to all wells. Incubate, with shaking, for 10minutes.

12. Stop reaction by adding 25 μl 500 mM EDTA (pH 8.0) to all wells.

13. Wash as in step 4.

14. Add 100 μL HRP conjugated anti-Ptyr diluted 1:6000 in AntibodyDilution Buffer to each well. Incubate, with shaking, for 1 hr. at roomtemperature.

15. Wash plate 3× with TBST and 1× with PBS.

16. Add 100 μL of ABST solution to each well.

17. If necessary, stop the development reaction by adding 20 μL 10% SDSto each well.

18. Read plate on ELISA reader with test filter at 410 nM and referencefilter at 630 nM.

FGFR1 Bioassay

This assay is used to measure the in vitro kinase activity of FGF1-R inan ELISA assay.

Materials and Reagents

1. Costar 96-well ELISA plates (Corning Catalog #3369).

2. Poly(Glu-Tyr) (Sigma Catalog #PO275).

3. PBS (Gibco Catalog #450-1300EB)

4. 50 mM Hepes Buffer Solution.

5. Blocking Buffer (5% BSA/PBS).

6. Purified GST-FGFR1 (SUGEN, Inc.)

7. Kinase Dilution Buffer. Mix 500 μl 1M Hepes (GIBCO), 20 μl 5%BSA/PBS, 10 μl 100 mM sodium orthovanadate and 50 μl 5M NaCl.

8. 10 mM ATP

9. ATP/MnCl₂ phosphorylation mix: mix 20 μL ATP, 400 μL 1M MnCl₂ and9.56 ml dH₂O.

10. NUNC 96-well V bottom polypropylene plates (Applied ScientificCatalog #AS-72092).

11. 0.5M EDTA.

12. 0.05% TBST Add 500 μL TWEEN to 1 liter TBS.

13. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN, Inc.).

14. Goat anti-rabbit IgG peroxidase conjugate (Biosource, Catalog#ALI0404).

15. ABTS Solution.

16. ABTS/H₂O₂ solution.

Procedure

1. Coat Costar 96 well ELISA plates with 1 μg per well Poly(Glu-Tyr) in100 μl PBS. Store overnight at 4° C.

2. Wash coated plates once with PBS.

3. Add 150 μL of 5% BSA/PBS Blocking Buffer to each well. Incubate, withshaking, for 1 hr at room temperature.

4. Wash plate 2× with PBS, then once with 50 mM Hepes. Pat plates on apaper towel to remove excess liquid and bubbles.

5. Add 25 μL of 0.4 mM test compound in 4% DMSO or 4% DMSO alone(controls) to plate.

6. Dilute purified GST-FGFR1 in Kinase Dilution Buffer (5 ng kinase/50ul KDB/well).

7. Add 50 μL of diluted kinase to each well.

8. Start kinase reaction by adding 25 μl/well of freshly preparedATP/Mn++ (0.4 ml 1M MnCl₂, 40 μL 10 mM ATP, 9.56 ml dH₂O), freshlyprepared).

9. Stop reaction with 25 μL of 0.5M EDTA.

10. Wash plate 4× with fresh TBST.

11. Make up Antibody Dilution Buffer: For 50 ml, mix 5 ml of 5% BSA, 250μl of 5% milk and 50 μl of 100 mM sodium vanadate, bring to final volumewith 0.05% TBST.

12. Add 100 μl per well of anti-phosphotyrosine (1:10000 dilution inADB). Incubate, with shaking for 1 hr. at room temperature.

13. Wash as in step 10.

14. Add 100 μl per well of Biosource Goat anti-rabbit IgG peroxidaseconjugate (1:6000 dilution in ADB). Incubate, with shaking for 1 hr. atroom temperature.

15. Wash as in step 10 and then with PBS to remove bubbles and excessTWEEN.

16. Add 100 μl of ABTS/H₂O₂ solution to each well.

17. Incubate, with shaking, for 10 to 20 minutes. Remove any bubbles.

18. Read assay on Dynatech MR7000 ELISA reader: test filter at 410 nM,reference filter at 630 nM.

EGFR Bioassay

This assay is used to the in vitro kinase activity of EGFR in an ELISAassay.

Materials and Reagents

1. Corning 96-well ELISA plates.

2. SUMO1 monoclonal anti-EGFR antibody (SUGEN, Inc.).

3. PBS.

4. TBST Buffer.

5. Blocking Buffer: for 100 ml, mix 5.0 g Carnation® Instant Non-fatMilk with 100 ml of PBS.

6. A431 cell lysate (SUGEN, Inc.).

7. TBS Buffer.

8. TBS+10% DMSO: for 1L, mix 1.514 g TRIS, 2.192 g NaCl and 25 ml DMSO;bring to 1 liter total volume with dH₂O.

9. ATP (Adenosine-5′-triphosphate, from Equine muscle, Sigma Cat. No.A-5394), 1.0 mM solution in dH₂O. This reagent should be made upimmediately prior to use and kept on ice.

10. 1.0 mM MnCl₂.

11. ATP/MnCl₂ phosphorylation mix: for 10 ml, mix 300 μl of 1 mM ATP,500 μl MnCl₂ and 9.2 ml dH₂O. Prepare just prior to use, keep on ice.

12. NUNC 96-well V bottom polypropylene plates.

13. EDTA.

14. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN, Inc.).

15. Goat anti-rabbit IgG peroxidase conjugate (Biosource Cat. No.ALI0404).

16. ABTS.

17. 30% Hydrogen peroxide.

18. ABTS/H₂O₂.

19. 0.2 M HCl.

Procedure

1. Coat Corning 96 well ELISA plates with 0.5 μg SUMO1 in 100 μl PBS perwell, hold overnight at 4° C.

2. Remove unbound SUMO1 from wells by inverting plate to remove liquid.Wash 1× with dH₂O. Pat the plate on a paper towel to remove excessliquid.

3. Add 150 μl of Blocking Buffer to each well. Incubate, with shaking,for 30 min. at room temperature.

4. Wash plate 3× with deionized water, then once with TBST. Pat plate ona paper towel to remove excess liquid and bubbles.

5. Dilute lysate in PBS (7 μg lysate/100 μl PBS).

6. Add 100 μl of diluted lysate to each well. Shake at room temperaturefor 1 hr.

7. Wash plates as in 4, above.

8. Add 120 μl TBS to ELISA plate containing captured EGFR.

9. Dilute test compound 1:10 in TBS, place in well

10. Add 13.5 μl diluted test compound to ELISA plate. To control wells,add 13.5 μl TBS in 10% DMSO.

11. Incubate, with shaking, for 30 minutes at room temperature.

12. Add 15 μl phosphorylation mix to all wells except negative controlwell. Final well volume should be approximately 150 μl with 3 μM ATP/5mM MnCl₂ final concentration in each well. Incubate with shaking for 5minutes.

13. Stop reaction by adding 16.5 μl of EDTA solution while shaking.Shake for additional 1 min.

14. Wash 4× with deionized water, 2× with TBST.

15. Add 100 μl anti-phosphotyrosine (1:3000 dilution in TBST) per well.Incubate, with shaking, for 30-45 min. at room temperature.

16. Wash as in 4, above.

17. Add 100 μl Biosource Goat anti-rabbit IgG peroxidase conjugate(1:2000 dilution in TBST) to each well. Incubate with shaking for 30min. at room temperature.

18. Wash as in 4, above.

19. Add 100 μl of ABTS/H₂O₂ solution to each well.

20. Incubate 5 to 10 minutes with shaking. Remove any bubbles.

21. If necessary, stop reaction by adding 100 μl 0.2 M HCl per well.

22. Read assay on Dynatech MR7000 ELISA reader: test filter at 410 nM,reference filter at 630 nM.

This assay is used to the in vitro kinase activity of PDGFR in an ELISAassay.

Materials and Reagents

1. Corning 96-well ELISA plates

2. 28D4C10 monoclonal anti-PDGFR antibody (SUGEN, Inc.).

3. PBS.

4. TBST Buffer.

5. Blocking Buffer (same as for EGFR bioassay).

6. PDGFR-β expressing NIH 3T3 cell lysate (SUGEN, Inc.).

7. TBS Buffer.

8. TBS+10% DMSO.

9. ATP.

10. MnCl₂.

11. Kinase buffer phosphorylation mix: for 10 ml, mix 250 μl 1M TRIS,200 μl 5M NaCl, 100 μl 1M MnCl₂ and 50 μl 100 mM Triton X-100 in enoughdH₂O to make 10 ml.

12. NUNC 96-well V bottom polypropylene plates.

13. EDTA.

14. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN, Inc.).

15. Goat anti-rabbit IgG peroxidase conjugate (Biosource Cat. No.ALI0404).

16. ABTS.

17. Hydrogen peroxide, 30% solution.

18. ABTS/H₂O₂.

19. 0.2M HCl.

Procedure

1. Coat Corning 96 well ELISA plates with 0.5 μg 28D4C10 in 100 μl PBSper well, hold overnight at 4° C.

2. Remove unbound 28D4C10 from wells by inverting plate to removeliquid. Wash 1× with dH₂O. Pat the plate on a paper towel to removeexcess liquid.

3. Add 150 μl of Blocking Buffer to each well. Incubate for 30 min. atroom temperature with shaking.

4. Wash plate 3× with deionized water, then once with TBST. Pat plate ona paper towel to remove excess liquid and bubbles.

5. Dilute lysate in HNTG (10 μg lysate/100 μl HNTG).

6. Add 100 μl of diluted lysate to each well. Shake at room temperaturefor 60 min.

7. Wash plates as described in Step 4.

8. Add 80 μl working kinase buffer mix to ELISA plate containingcaptured PDGFR.

9. Dilute test compound 1:10 in TBS in 96-well polypropylene plates.

10. Add 10 μl diluted test compound to ELISA plate. To control wells,add 10 μl TBS+10% DMSO. Incubate with shaking for 30 minutes at roomtemperature.

11. Add 10 μl ATP directly to all wells except negative control well(final well volume should be approximately 100 μl with 20 μM ATP in eachwell.) Incubate 30 minutes with shaking.

12. Stop reaction by adding 10 μl of EDTA solution to each well.

13. Wash 4× with deionized water, twice with TBST.

14. Add 100 μl anti-phosphotyrosine (1:3000 dilution in TBST) per well.Incubate with shaking for 30-45 min. at room temperature.

15. Wash as in Step 4.

16. Add 100 μl Biosource Goat anti-rabbit IgG peroxidase conjugate(1:2000 dilution in TBST) to each well. Incubate with shaking for 30min. at room temperature.

17. Wash as in Step 4.

18. Add 100 μl of ABTS/H₂O₂ solution to each well.

19. Incubate 10 to 30 minutes with shaking. Remove any bubbles.

20. If necessary stop reaction with the addition of 100 μl 0.2 M HCl perwell.

21. Read assay on Dynatech MR7000 ELISA reader with test filter at 410nM and reference filter at 630 nM.

Cellular HER-2 Kinase Assay

This assay is used to measure HER-2 kinase activity in whole cells in anELISA format.

Materials and Reagents

1. DMEM (GIBCO Catalog #11965-092).

2. Fetal Bovine Serum (FBS, GIBCO Catalog #16000-044), heat inactivatedin a water bath for 30 min. at 56° C.

3. Trypsin (GIBCO Catalog #25200-056).

4. L-Glutamine (GIBCO Catalog #25030-081).

5. HEPES (GIBCO Catalog #15630-080).

6. Growth Media: Mix 500 ml DMEM, 55 ml heat inactivated FBS, 10 mlHEPES and 5.5 ml L-Glutamine.

7. Starve Media: Mix 500 ml DMEM, 2.5 ml heat inactivated FBS, 10 mlHEPES and 5.5 ml L-Glutamine.

8. PBS.

9. Flat Bottom 96-well Tissue Culture Micro Titer Plates (CorningCatalog #25860).

10. 15 cm Tissue Culture Dishes (Corning Catalog #08757148).

11. Coming 96-well ELISA Plates.

12. NUNC 96-well V bottom polypropylene plates.

13. Costar Transfer Cartridges for the Transtar 96 (Costar Catalog#7610).

14. SUMO 1: monoclonal anti-EGFR antibody (SUGEN, Inc.).

15. TBST Buffer.

16. Blocking Buffer: 5% Carnation Instant Milk® in PBS.

17. EGF Ligand: EGF-201, Shinko American, Japan. Suspend powder in 100μL of 10 mM HCl. Add 10 μL 10 mM NaOH. Add 800 μL PBS and transfer to anEppendorf tube, store at −20° C. until ready to use.

18. HNTG Lysis Buffer: For Stock 5×HNTG, mix 23.83 g Hepes, 43.83 gNaCl, 500 ml glycerol and 100 ml Triton X-100 and enough dH₂O to make 1L of total solution. For 1×HNTG*, mix 2 ml 5× HNTG, 100 μL 0.1M Na₃VO₄,250 μL 0.2M Na₄P₂O₇ and 100 μL EDTA.

19. EDTA.

20. Na₃VO₄: To make stock solution, mix 1.84 g Na₃VO₄ with 90 ml dH2O.Adjust pH to 10. Boil in microwave for one minute (solution becomesclear). Cool to room temperature. Adjust pH to 10. Repeatheating/cooling cycle until pH remains at 10.

21. 200 mM Na₄P₂O₇.

22. Rabbit polyclonal antiserum specific for phosphotyrosine (anti-Ptyrantibody, SUGEN, Inc.).

23. Affinity purified antiserum, goat anti-rabbit IgG antibody,peroxidase conjugate (Biosource Cat #ALI0404).

24. ABTS Solution.

25. 30% Hydrogen peroxide solution.

26. ABTS/H₂O₂.

27. 0.2 M HCl.

Procedure

1. Coat Coming 96 well ELISA plates with SUMO1 at 1.0 μg per well inPBS, 100 μl final volume/well. Store overnight at 4° C.

2. On day of use, remove coating buffer and wash plate 3 times with dH₂Oand once with TBST buffer. All washes in this assay should be done inthis manner, unless otherwise specified.

3. Add 100 μL of Blocking Buffer to each well. Incubate plate, withshaking, for 30 min. at room temperature. Just prior to use, wash plate.

4. Use EGFr/HER-2 chimera/3T3-C7 cell line for this assay.

5. Choose dishes having 80-90% confluence. Collect cells bytrypsinization and centrifuge at 1000 rpm at room temperature for 5 min.

6. Resuspend cells in starve medium and count with trypan blue.Viability above 90% is required. Seed cells in starve medium at adensity of 2,500 cells per well, 90 μL per well, in a 96 well microtiterplate. Incubate seeded cells overnight at 37° under 5% CO₂.

7. Start the assay two days after seeding.

8. Test compounds are dissolved in 4% DMSO. Samples are then furtherdiluted directly on plates with starve-DMEM. Typically, this dilutionwill be 1:10 or greater. All wells are then transferred to the cellplate at a further 1:10 dilution (10 μl sample and media into 90 μl ofstarve media). The final DMSO concentration should be 1% or lower. Astandard serial dilution may also be used.

9. Incubate under 5% CO₂ at 37° C. for 2 hours.

10. Prepare EGF ligand by diluting stock EGF (16.5 FM) in warm DMEM to150 nM.

11. Prepare fresh HNTG* sufficient for 100 μL per well; place on ice.

12. After 2 hour incubation with test compound, add prepared EGF ligandto cells, 50 μL per well, for a final concentration of 50 nM. Positivecontrol wells receive the same amount of EGF. Negative controls do notreceive EGF. Incubate at 37° C. for 10 min.

13. Remove test compound, EGF, and DMEM. Wash cells once with PBS.

14. Transfer HNTG* to cells, 100 μL per well. Place on ice for 5minutes. Meanwhile, remove blocking buffer from ELISA plate and wash.

15. Scrape cells from plate with a micropipettor and homogenize cellmaterial by repeatedly aspirating and dispensing the HNTG* lysis buffer.Transfer lysate to a coated, blocked, washed ELISA plate.

16. Incubate, with shaking, at room temperature for 1 hr.

17. Remove lysate, wash. Transfer freshly diluted anti-Ptyr antibody(1:3000 in TBST) to ELISA plate, 100 μL per well.

18. Incubate, with shaking, at room temperature, for 30 min.

19. Remove anti-Ptyr antibody, wash. Transfer freshly diluted BIOSOURCEantibody to ELISA plate(1:8000 in TBST, 100 μL per well).

20. Incubate, with shaking, at room temperature for 30 min.

21. Remove BIOSOURCE antibody, wash. Transfer freshly prepared ABTS/H₂O₂solution to ELISA plate, 100 μL per well.

22. Incubate, with shaking, for 5-10 minutes. Remove any bubbles.

23. Stop reaction by adding 100 μL of 0.2M HCl per well.

24. Read assay on Dynatech MR7000 ELISA reader with test filter set at410 nM and reference filter at 630 nM.

CDK2/Cyclin A Assay

This assay is used to measure the in vitro serine/threonine kinaseactivity of human cdk2/cyclin A in a Scintillation Proximity Assay(SPA).

Materials and Reagents

1. Wallac 96-well polyethylene terephthalate (flexi) plates (WallacCatalog #1450-401).

2. Amersham Redivue [γ³³P] ATP (Amersham catalog #AH9968).

3. Amersham streptavidin coated polyvinyltoluene SPA beads (Amershamcatalog #RPNQ0007). The beads should be reconstituted in PBS withoutmagnesium or calcium, at 20 mg/ml.

4. Activated cdk2/cyclin A enzyme complex purified from Sf9 cells(SUGEN, Inc.).

5. Biotinylated peptide substrate (Debtide). Peptide biotin-X-PKTPKKAKKLis dissolved in dH₂O at a concentration of 5 mg/ml.

6. 20% DMSO in dH₂O.

7. Kinase buffer: for 10 ml, mix 9.1 ml dH₂O, 0.5 ml TRIS(pH 7.4), 0.2ml 1M MgCl₂, 0.2 ml 10% NP40 and 0.02 ml 1M DTT, added fresh just priorto use.

8. 10 mM ATP in dH₂O.

9. 1M Tris, pH adjusted to 7.4 with HCl.

10. 1M MgCl₂.

11. 1MDTT.

12. PBS (Gibco Catalog #14190-144).

13. 0.5M EDTA.

14. Stop solution: For 10 ml, mix 9.25 ml PBS, 0.05 ml 10 mM ATP, 0.1 ml0.5 M EDTA, 0.1 ml 10% Triton X-100 and 1.5 ml of 50 mg/ml SPA beads.

Procedure

1. Prepare solutions of test compounds at 4×the desired finalconcentration in 5% DMSO. Add 10 μL to each well. For positive andnegative controls, use 10 μL 20% DMSO alone in wells.

2. Dilute the peptide substrate (deb-tide) 1:250 with dH2O to give afinal concentration of 0.02 mg/ml.

3. Mix 24 μL 0.1 mM ATP with 24 μCi γ³³P ATP and enough dH₂O to make 600μL.

4. Mix diluted peptide and ATP solutions 1:1 (600 μL+600 μL per plate).Add 10 μL of this solution to each well.

5. Dilute 5 μL of cdk2/cyclin A solution into 2.1 ml 2×kinase buffer(per plate). Add 20 μL enzyme per well. For negative controls, add 20 μL2×kinase buffer without enzyme.

6. Mix briefly on a plate shaker; incubate for 60 minutes.

7. Add 200 μL stop solution per well.

8. Let stand at least 10 min.

9. Spin plate at approx. 2300 rpm for 10-15 min.

10. Count plate on Trilux reader.

Met Transphosphorylation Assay

This assay is used to measure phosphotyrosine levels on a poly(glutamicacid:tyrosine, 4:1) substrate as a means for identifyingagonists/antagonists of met transphosphorylation of the substrate.

Materials and Reagents

1. Corning 96-well ELISA plates, Corning Catalog #25805-96.

2. Poly(glu-tyr), 4:1, Sigma, Cat. No; P 0275.

3. PBS, Gibco Catalog #450-1300EB

4. 50 mM HEPES

5. Blocking Buffer: Dissolve 25 g Bovine Serum Albumin, Sigma Cat. NoA-7888, in 500 ml PBS, filter through a 4 μm filter.

6. Purified GST fusion protein containing the Met kinase domain, SUGEN,Inc.

7. TBST Buffer.

8. 10% aqueous (MilliQue H₂O) DMSO.

9. 10 mM aqueous (dH₂O) Adenosine-5′-triphosphate, Sigma Cat. No.A-5394.

10. 2×Kinase Dilution Buffer: for 100 ml, mix 10 mL 1M HEPES at pH 7.5with 0.4 mL 5% BSA/PBS, 0.2 mL 0.1 M sodium orthovanadate and 1 mL 5Msodium chloride in 88.4 mL dH₂O.

11. 4×ATP Reaction Mixture: for 10 mL, mix 0.4 mL 1 M manganese chlorideand 0.02 mL 0.1 M ATP in 9.56 mL dH₂O.

12. 4×Negative Controls Mixture: for 10 mL, mix 0.4 mL 1 M manganesechloride in 9.6 mL dH₂O.

13. NUNC 96-well V bottom polypropylene plates, Applied ScientificCatalog #S-72092

14. 500 mM EDTA.

15. Antibody Dilution Buffer: for 100 mL, mix 10 mL 5% BSA/PBS, 0.5 mL5% Carnation® Instant Milk in PBS and 0.1 mL 0.1 M sodium orthovanadatein 88.4 mL TBST.

16. Rabbit polyclonal antophosphotyrosine antibody, SUGEN, Inc.

17. Goat anti-rabbit horseradish peroxidase conjugated antibody,Biosource, Inc.

18. ABTS Solution: for 1 L, mix 19.21 g citric acid, 35.49 g Na₂HPO₄ and500 mg ABTS with sufficient dH₂O to make 1 L.

19. ABTS/H₂O₂: mix 15 mL ABST solution with 2 μL H₂O₂ five minutesbefore use.

20. 0.2 M HCl

Procedure

1. Coat ELISA plates with 2 μg Poly(Glu-Tyr) in 100 μL PBS, holdovernight at 4° C.

2. Block plate with 150 μL of 5% BSA/PBS for 60 min.

3. Wash plate twice with PBS then once with 50 mM Hepes buffer pH 7.4.

4. Add 50 μl of the diluted kinase to all wells. (Purified kinase isdiluted with Kinase Dilution Buffer. Final concentration should be 10ng/well.)

5. Add 25 μL of the test compound (in 4% DMSO) or DMSO alone (4% indH₂O) for controls to plate.

6. Incubate the kinase/compound mixture for 15 minutes.

7. Add 25 μL of 40 mM MnCl₂ to the negative control wells.

8. Add 25 μL ATP/MnCl₂ mixture to the all other wells (except thenegative controls). Incubate for 5 min.

9. Add 25 μL 500 mM EDTA to stop reaction.

10. Wash plate 3× with TBST.

11. Add 100 μL rabbit polyclonal anti-Ptyr diluted 1:10,000 in AntibodyDilution Buffer to each well. Incubate, with shaking, at roomtemperature for one hour.

12. Wash plate 3× with TBST.

13. Dilute Biosource HRP conjugated anti-rabbit antibody 1:6,000 inAntibody Dilution buffer. Add 100 μL per well and incubate at roomtemperature, with shaking, for one hour.

14. Wash plate 1× with PBS.

15. Add 100 μl of ABTS/H₂O₂ solution to each well.

16. If necessary, stop the development reaction with the addition of 100μl of 0.2M HCl per well.

17. Read plate on Dynatech MR7000 ELISA reader with the test filter at410 nM and the reference filter at 630 nM.

IGF-1 Transphosphorylation Assay

This assay is used to measure the phosphotyrosine level in poly(glutamicacid:tyrosine, 4:1) for the identification of agonists/antagonists ofgst-IGF-1 transphosphorylation of a substrate.

Materials and Reagents

1. Corning 96-well ELISA plates.

2. Poly(Glu-Tyr),4:1, Sigma Cat. No. P0275.

3. PBS, Gibco Catalog #450-1300EB.

4. 50 mM HEPES

5. TBB Blocking Buffer: for 1 L, mix 100 g BSA, 12.1 gTRIS (pH 7.5),58.44 g sodium chloride and 10 mL 1% TWEEN-20.

6. Purified GST fusion protein containing the IGF-1 kinase domain(SUGEN, Inc.)

7. TBST Buffer: for 1 L, mix 6.057 g Tris, 8.766 g sodium chloride and0.5 ml TWEEN-20 with enough dH₂O to make 1 liter.

8. 4% DMSO in Milli-Q H₂O.

9. 10 mM ATP in dH₂O.

10. 2×Kinase Dilution Buffer: for 100 mL, mix 10 mL 1 M HEPES (pH 7.5),0.4 mL 5% BSA in dH₂O, 0.2 mL 0.1 M sodium orthovanadate and 1 mL 5 Msodium chloride with enough dH₂O to make 100 mL.

11. 4×ATP Reaction Mixture: for 10 mL, mix 0.4 mL 1 M MnCl₂ and 0.008 mL0.01 M ATP and 9.56 mL dH₂O.

12. 4×Negative Controls Mixture: mix 0.4 mL 1 M MnCl₂ in 9.60 mL dH₂O.

13. NUNC 96-well V bottom polypropylene plates.

14. 500 mM EDTA in dH₂O.

15. Antibody Dilution Buffer: for 100 mL, mix 10 mL 5% BSA in PBS, 0.5mL 5% Carnation Instant Non-fat Milk in PBS and 0.1 mL 0.1 M sodiumorthovanadate in 88.4 mL TBST.

16. Rabbit Polyclonal antiphosphotyrosine antibody, SUGEN, Inc.

17. Goat anti-rabbit HRP conjugated antibody, Biosource.

18. ABTS Solution.

20. ABTS/H₂O₂: mix 15 mL ABTS with 2 μL H₂O₂, 5 minutes before using.

21. 0.2 M HCl in dH₂O.

Procedure

1. Coat ELISA plate with 2.0 μg/well Poly(Glu, Tyr), 4:1 (Sigma P0275)in 100 μl PBS. Store plate overnight at 4° C.

2. Wash plate once with PBS.

3. Add 100 μl of TBB Blocking Buffer to each well. Incubate plate for 1hour with shaking at room temperature.

4. Wash plate once with PBS, then twice with 50 mM Hepes buffer pH 7.5.

5. Add 25 μL of test compound in 4% DMSO (obtained by diluting a stocksolution of 10 mM test compound in 100% DMSO with dH₂O) to plate.

6. Add 10.0 ng of gst-IGF-1 kinase in 50 μl Kinase Dilution Buffer toall wells.

7. Start kinase reaction by adding 25 μl 4×ATP Reaction Mixture to alltest wells and positive control wells. Add 25 μl 4×Negative ControlsMixture to all negative control wells. Incubates for 10 minutes, withshaking, at room temperature.

8. Add 25 μl 0.5M EDTA (pH 8.0) to all wells.

9. Wash plate 4× with TBST Buffer.

10. Add rabbit polyclonal anti-phosphotyrosine antisera at a dilution of1:10,000 in 100 μl Antibody Dilution Buffer to all wells. Incubate, withshaking, at room temperature for 1 hour.

11. Wash plate as in step 9.

12. Add 100 μL Biosource anti-rabbit HRP at a dilution of 1:10,000 inAntibody dilution buffer to all wells. Incubate, with shaking, at roomtemperature for 1 hour.

13. Wash plate as in step 9, follow with one wash with PBS to removebubbles and excess Tween-20.

14. Develop by adding 100 μl/well ABTS/H₂O₂ to each well

15. After about 5 minutes, read on ELISA reader with test filter at 410nm and referenced filter at 630 nm.

BrdU Incorporation Assays

The following assays use cells engineered to express a selected receptorand then evaluate the effect of a compound of interest on the activityof ligand-induced DNA synthesis by determining BrdU incorporation intothe DNA.

The following materials, reagents and procedure are general to each ofthe following BrdU incorporation assays. Variances in specific assaysare noted.

General Materials and Reagents

1. The appropriate ligand.

2. The appropriate engineered cells.

3. BrdU Labeling Reagent: 10 mM, in PBS, pH7.4(Roche MolecularBiochemicals, Indianapolis, Ind.).

4. FixDenat: fixation solution (Roche Molecular Biochemicals,Indianapolis, Ind.).

5. Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase(Chemicon, Temecula, Calif.).

6. TMB Substrate Solution: tetramethylbenzidine (TMB, ready to use,Roche Molecular Biochemicals, Indianapolis, Ind.).

7. PBS Washing Solution: 1×PBS, pH 7.4.

8. Albumin, Bovine (BSA), fraction V powder (Sigma Chemical Co., USA).

General Procedure

1. Cells are seeded at 8000 cells/well in 10% CS, 2 mM Gln in DMEM, in a96 well plate. Cells are incubated overnight at 37° C. in 5% CO₂.

2. After 24 hours, the cells are washed with PBS, and then areserum-starved in serum free medium (0% CS DMEM with 0.1% BSA) for 24hours.

3. On day 3, the appropriate ligand and the test compound are added tothe cells simultaneously. The negative control wells receive serum freeDMEM with 0.1% BSA only; the positive control cells receive the ligandbut no test compound. Test compounds are prepared in serum free DMEMwith ligand in a 96 well plate, and serially diluted for 7 testconcentrations.

4. After 18 hours of ligand activation, diluted BrdU labeling reagent(1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU(final concentration is 10 μM) for 1.5 hours.

5. After incubation with labeling reagent, the medium is removed bydecanting and tapping the inverted plate on a paper towel. FixDenatsolution is added (50 μl/well) and the plates are incubated at roomtemperature for 45 minutes on a plate shaker.

6. The FixDenat solution is removed by decanting and tapping theinverted plate on a paper towel. Milk is added (5% dehydrated milk inPBS, 200 μl/well) as a blocking solution and the plate is incubated for30 minutes at room temperature on a plate shaker.

7. The blocking solution is removed by decanting and the wells arewashed once with PBS. Anti-BrdU-POD solution is added (1:200 dilution inPBS, 1% BSA, 50 μl/well) and the plate is incubated for 90 minutes atroom temperature on a plate shaker.

8. The antibody conjugate is removed by decanting and rinsing the wells5 times with PBS, and the plate is dried by inverting and tapping on apaper towel.

9. TMB substrate solution is added (100 μl/well) and incubated for 20minutes at room temperature on a plate shaker until color development issufficient for photometric detection.

10. The absorbance of the samples are measured at 410 nm (in “dualwavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

EGF-Induced BrdU Incorporation Assay

Materials and Reagents

1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).

2. 3T3/EGFRc7.

Remaining Materials and Reagents and Procedure, as above.

EGF-Induced Her-2-driven BrdU Incorporation Assay

Materials and Reagents

1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).

2. 3T3/EGFr/Her2/EGFr (EGFr with a Her-2 kinase domain).

Remaining Materials and Reagents and Procedure, as above.

EGF-Induced Her-4-driven BrdU Incorporation Assay

Materials and Reagents

1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).

2. 3T3/EGFr/Her4/EGFr (EGFr with a Her-4 kinase domain).

Remaining Materials and Reagents and Procedure, as above.

PDGF-Induced BrdU Incorporation Assay

Materials and Reagents

1. Human PDGF B/B (Boehringer Mannheim, Germany).

2. 3T3/EGFRc7.

Remaining Materials and Reagents and Procedure, as above.

FGF-Induced BrdU Incorporation Assay

Materials and Reagents

1. Human FGF2/bFGF (Gibco BRL, USA).

2. 3T3c7/EGFr

Remaining Materials and Reagents and Procedure, as above.

IGF1-Induced BrdU Incorporation Assay

Materials and Reagents

1. Human, recombinant (G511, Promega Corp., USA)

2. 3T3/IGF1r.

Remaining Materials and Reagents and Procedure, as above.

Insulin-Induced BrdU Incorporation Assay

Materials and Reagents

1. Insulin, crystalline, bovine, Zinc (13007, Gibco BRL, USA).

2. 3T3/H25.

Remaining Materials and Reagents and Procedure, as above.

HGF-Induced BrdU Incorporation Assay

Materials and Reagents

1. Recombinant human HGF (Cat. No. 249-HG, R&D Systems, Inc. USA).

2. B×PC-3 cells (ATCC CRL-1687).

Remaining Materials and Reagents, as above.

Procedure

1. Cells are seeded at 9000 cells/well in RPMI 10% FBS in a 96 wellplate. Cells are incubated overnight at 37° C. in 5% CO₂.

2. After 24 hours, the cells are washed with PBS, and then are serumstarved in 100 μl serum-free medium (RPMI with 0.1% BSA) for 24 hours.

3. On day 3, 25 μl containing ligand (prepared at 1 μg/ml in RPMI with0.1% BSA; final HGF conc. is 200 ng/ml) and test compounds are added tothe cells. The negative control wells receive 25 μl serum-free RPMI with0.1% BSA only; the positive control cells receive the ligand (HGF) butno test compound. Test compounds are prepared at 5 times their finalconcentration in serum-free RPMI with ligand in a 96 well plate, andserially diluted to give 7 test concentrations. Typically, the highestfinal concentration of test compound is 100 μM, and 1:3 dilutions areused (i.e. final test compound concentration range is 0.137-100 μM).

4. After 18 hours of ligand activation, 12.5 μl of diluted BrdU labelingreagent (1:100 in RPMI, 0.1% BSA) is added to each well and the cellsare incubated with BrdU (final concentration is 10 μM) for 1 hour.

5. Same as General Procedure.

6. Same as General Procedure.

7. The blocking solution is removed by decanting and the wells arewashed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1%BSA) is added (100 μl/well) and the plate is incubated for 90 minutes atroom temperature on a plate shaker.

8. Same as General Procedure.

9. Same as General Procedure.

10. Same as General Procedure.

Exponential BrdU Incorporation Assay

This assay is used to measure the proliferation (DNA synthesis) ofexponentially growing A431 cells. The assay will screen for compoundsthat inhibit cell cycle progression.

Materials and Reagents

Healthy growing A431 cells. The remainder of the Materials and Reagentsare the same as listed above in the general protocol section.

Procedure

1. A431 cells are seeded at 8000 cells/well in 10% FBS, 2 mM Gln inDMEM, on a 96-well plate. Cells are incubated overnight at 37° C. in 5%CO₂.

2. On day 2, test compounds are serially diluted to 7 testconcentrations in the same growth medium on a 96-well plate and then areadded to the cells on a 96-well tissue culture plate.

3. After 20-24 hours of incubation, diluted BrdU labeling reagent (1:100in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU (finalconcentration is 10 μM) for 2 hours.

Steps 5-10 of the General Procedure are used to complete the assay.

ZenSrc Assay

This assay is used to screen for inhibitors of the tyrosine kinase Src.

Materials and Reagents

1. Coating buffer: PBS containing sodium azide (0.2 mg/ml).

2.1% w/v BSA in PBS.

3. Wash buffer: PBS containing 0.05% v/v Tween 20 (PBS-TWEEN)

4. 500 mM HEPES pH7.4.

5. ATP (40 μM)+MgCl₂ (80 mM) in distilled water.

6. MgCl₂ (80 mM) in distilled water (for no ATP blanks).

7. Test compounds, 10 mM in DMSO.

8. Assay Buffer: 100 mM HEPES, pH 7.4, containing 2 mM DTT, 0.2 mMsodium orthovanadate and 0.2 mgs/ml BSA.

9. Partially purified recombinant human Src (UBI (14-117)

10. Anti-phosphotyrosine (SUGEN rabbit polyclonal anti-PY).

11. HRP-linked goat anti-rabbit Ig (Biosource International #6430)

12. HRP substrate ABTS or Pierce Peroxidase substrate.

13. Corning ELISA plates.

Procedure

1. Coat plates with 100 μl of 20 μg/ml poly(Glu-Tyr) (Sigma Cat. No.P0275) containing 0.01% sodium azide. Hold overnight at 4° C.

2. Block with 1% BSA at 100 μl/well for one hour at room temperature.

3. Plate test compounds (10 mM in DMSO) at 2 μl/well on a Costar plateready for dilution with dH₂O and plating to reaction plates.

4. Dilute Src kinase 1:10,000 in Reaction Buffer, for 5 plates prepare25 ml as follows: 2.5 mls 1M HEPES pH7.4 (stored sterile at 4° C.),21.85 ml distilled water, 0.1 ml 5% BSA, 0.5 ml 10 mM sodiumorthovanadate (stored sterile at 4° C.), 50 μl 1.0 M DTT (stored frozenat −20° C.), and 2.5 μl Src Kinase (stored frozen at −80° C.).

5. Add 48 μl of distilled water to the 2 μl of each compound in thedilution plate then add 25 μl/well of this to the reaction plate.

6. Add 50 μl of HRP to each reaction buffer well and then 25 μlATP-MgCl₂/well (MgCl₂ only to no ATP for 15 minutes on plate shaker.Stop reaction by adding 25 μl of 0.5M EDTA to each well.

7. Wash 4× with PBS-TWEEN.

8. Add 100 μl anti-phosphotyrosine (1:10,000 of anti-pTyr serum or1:3,000 of 10% glycerol diluted PA-affinity purified antibody) inPBS-TWEEN containing 0.5% BSA, 0.025% Non-fat milk powder and 100 μMsodium orthovanadate. Incubate with continuous shaking at roomtemperature for one hour.

9. Wash plates 4× with PBS-TWEEN.

10. Add 100 μl HRP-linked Ig (1:5,000) in PBS-TWEEN containing 0.5% BSA,0.025% Non-fat milk powder, 100 μM sodium orthovanadate. Incubate withshaking at room temperature for one hour.

11. Wash plates 4× with PBS-TWEEN and then once with PBS.

12. Develop plate using ABTS or other peroxidase substrate.

Cell Cycle Analysis

A431 cells in standard growth medium are exposed to a desiredconcentration of a test compound for 20-24 hours at 37° C. The cells arethen collected, suspended in PBS, fixed with 70% ice-cold methanol andstained with propidium iodide. The DNA content is then measured using aFACScan flow cytometer. Cell cycle phase distribution can then beestimated using CellFIT software (Becton-Dickinson).

HUV-EC-C Assay

This assay is used to measure a compound's activity against PDGF-R,FGF-R, VEGF, aFGF or Flk-1/KDR, all of which are naturally expressed byHUV-EC cells.

Day 0

1. Wash and trypsinize HUV-EC-C cells (human umbilical vein endothelialcells, (American Type Culture Collection, catalogue no. 1730 CRL). Washwith Dulbecco's phosphate-buffered saline (D-PBS, obtained from GibcoBRL, catalogue no. 14190-029) 2 times at about 1 ml/10 cm² of tissueculture flask. Trypsinize with 0.05% trypsin-EDTA in non-enzymatic celldissociation solution (Sigma Chemical Company, catalogue no. C-1544).The 0.05% trypsin is made by diluting 0.25% trypsin/1 mM EDTA (Gibco,catalogue no. 25200-049) in the cell dissociation solution. Trypsinizewith about 1 ml/25-30 cm² of tissue culture flask for about 5 minutes at37° C. After cells have detached from the flask, add an equal volume ofassay medium and transfer to a 50 ml sterile centrifuge tube (FisherScientific, catalogue no. 05-539-6).

2. Wash the cells with about 35 ml assay medium in the 50 ml sterilecentrifuge tube by adding the assay medium, centrifuge for 10 minutes atapproximately 200× g, aspirate the supernatant, and resuspend with 35 mlD-PBS. Repeat the wash two more times with D-PBS, resuspend the cells inabout 1 ml assay medium/15 cm² of tissue culture flask. Assay mediumconsists of F12K medium (Gibco BRL, catalogue no. 21127-014) and 0.5%heat-inactivated fetal bovine serum. Count the cells with a CoulterCounter® (Coulter Electronics, Inc.) and add assay medium to the cellsto obtain a concentration of 0.8-1.0×10⁵ cells/ml.

3. Add cells to 96-well flat-bottom plates at 100 μl/well or 0.8-1.0×10⁴cells/well, incubate ˜24 h at 37° C., 5% CO₂.

Day 1

1. Make up two-fold test compound titrations in separate 96-well plates,generally 50 μM on down to 0 μM. Use the same assay medium as mentionedin day 0, step 2 above. Titrations are made by adding 90 μl/well of testcompound at 200 μM (4×the final well concentration) to the top well of aparticular plate column. Since the stock test compound is usually 20 mMin DMSO, the 200 μM drug concentration contains 2% DMSO.

A diluent made up to 2% DMSO in assay medium (F12K+0.5% fetal bovineserum) is used as diluent for the test compound titrations in order todilute the test compound but keep the DMSO concentration constant. Addthis diluent to the remaining wells in the column at 60 μl/well. Take 60μl from the 120 μl of 200 μM test compound dilution in the top well ofthe column and mix with the 60 μl in the second well of the column. Take60 μl from this well and mix with the 60 μl in the third well of thecolumn, and so on until two-fold titrations are completed. When thenext-to-the-last well is mixed, take 60 μl of the 120 μl in this welland discard it. Leave the last well with 60 μl of DMSO/media diluent asa non-test compound-containing control. Make 9 columns of titrated testcompound, enough for triplicate wells each for: (1) VEGF (obtained fromPepro Tech Inc., catalogue no. 100-200, (2) endothelial cell growthfactor (ECGF) (also known as acidic fibroblast growth factor, or AFGF)(obtained from Boehringer Mannheim Biochemica, catalogue no. 1439 600),or, (3) human PDGF B/B (1276-956, Boehringer Mannheim, Germany) andassay media control. ECGF comes as a preparation with sodium heparin.

2. Transfer 50 μl/well of the test compound dilutions to the 96-wellassay plates containing the 0.8-1.0×10⁴ cells/100 μl/well of theHUV-EC-C cells from day 0 and incubate 2 h at 37° C., 5% CO₂.

3. In triplicate, add 50 μl/well of 80 μg/ml VEGF, 20 ng/ml ECGF, ormedia control to each test compound condition. As with the testcompounds, the growth factor concentrations are 4×the desired finalconcentration. Use the assay media from day 0 step 2 to make theconcentrations of growth factors. Incubate approximately 24 hours at 37°C., 5% CO₂. Each well will have 50 μl test compound dilution, 50 μlgrowth factor or media, and 100 μl cells, which calculates to 200μl/well total. Thus the 4×concentrations of test compound and growthfactors become 1× once everything has been added to the wells.

Day 2

1. Add ³H-thymidine (Amersham, catalogue no. TRK-686) at 1 μCi/well (10μl/well of 100 μCi/ml solution made up in RPMI media+10%heat-inactivated fetal bovine serum) and incubate 24 h at 37° C., 5%CO₂. RPMI is obtained from Gibco BRL, catalogue no. 11875-051.

Day 3

1. Freeze plates overnight at −20° C.

Day 4

Thaw plates and harvest with a 96-well plate harvester (Tomtec Harvester96®) onto filter mats (Wallac, catalogue no. 1205-401), read counts on aWallac Betaplate™ liquid scintillation counter.

Vascular Permeability Assay

Increased vascular permeability in tumor-dependent angiogenesis is dueto a loosening of gap junctions in response to vascular endothelialgrowth factor (VEGF). The Miles assay for vascular permeability (Milesand Miles, J. Physiol. 118: 228-257 (1952)) has been adapted to athymicmice in order to evaluate the ability of the compounds of the presentinvention to inhibit VEGF-induced vascular permeability in vivo.

General Procedure

Test compound or vehicle is administered prior to (typically it is 4hours prior) to VEGF injection. 100 μl of 0.5% Evan's blue dye in PBS isinjected intravenously via lateral tail vein injections using a 27 gaugeneedle. Sixty minutes later, animals are anesthetized using the inhalantIsofluorane. Following anesthesia, VEGF (100 ng of VEGF in 20 μl of PBS)is injected intradermally in two spots and PBS (20 μl) is injected intwo spots in a grid pattern in the back of each animal. At a designatedtimepoint of up to 1 hour after VEGF injection, the animals areeuthanized by CO₂ and the skin patches are dissected and photographed.Based on a published report (Alicieri et al., Mol. Cell 4: 915-914(1999)) quantitative evaluation of the VEGF-dependent dye leakage intomouse skin can be achieved following elution of the dye from skinpatches.

In Vivo Animal Models

Xenograft Animal Models

The ability of human tumors to grow as xenografts in athymic mice (e.g.,Balb/c, nu/nu) provides a useful in vivo model for studying thebiological response to therapies for human tumors. Since the firstsuccessful xenotransplantation of human tumors into athymic mice,(Rygaard and Povlsen, 1969, Acta Pathol. Microbial. Scand. 77:758-760),nmany different human tumor cell lines (e.g., mammary, lung,genitourinary, gastro-intestinal, head and neck, glioblastoma, bone, andmalignant melanomas) have been transplanted and successfully grown innude mice. The following assays may be used to determine the level ofactivity, specificity and effect of the different compounds of thepresent invention. Three general types of assays are useful forevaluating compounds: cellular/catalytic, cellular/biological and invivo. The object of the cellular/catalytic assays is to determine theeffect of a compound on the ability of a TK to phosphorylate tyrosineson a known substrate in a cell. The object of the cellular/biologicalassays is to determine the effect of a compound on the biologicalresponse stimulated by a TK in a cell. The object of the in vivo assaysis to determine the effect of a compound in an animal model of aparticular disorder such as cancer.

Suitable cell lines for subcutaneous xenograft experiments include C6cells (glioma, ATCC # CCL 107), A375 cells (melanoma, ATCC # CRL 1619),A431 cells (epidermoid carcinoma, ATCC # CRL 1555), Calu 6 cells (lung,ATCC # HTB 56), PC3 cells (prostate, ATCC # CRL 1435), SKOV3TP5 cellsand NIH 3T3 fibroblasts genetically engineered to overexpress EGFR,PDGFR, IGF-1R or any other test kinase. The following protocol can beused to perform xenograft experiments:

Female athymic mice (BALB/c, nu/nu) are obtained from SimonsenLaboratories (Gilroy, Calif.). All animals are maintained underclean-room conditions in Micro-isolator cages with Alpha-dri bedding.They receive sterile rodent chow and water ad libitum.

Cell lines are grown in appropriate medium (for example, MEM, DMEM,Ham's F10, or Ham's F12 plus 5%-10% fetal bovine serum (FBS) and 2 mMglutamine (GLN)). All cell culture media, glutamine, and fetal bovineserum are purchased from Gibco Life Technologies (Grand Island, N.Y.)unless otherwise specified. All cells are grown in a humid atmosphere of90-95% air and 5-10% CO₂ at 37° C. All cell lines are routinelysubcultured twice a week and are negative for mycoplasma as determinedby the Mycotect method (Gibco).

Cells are harvested at or near confluency with 0.05% Trypsin-EDTA andpelleted at 450×g for 10 min. Pellets are resuspended in sterile PBS ormedia (without FBS) to a particular concentration and the cells areimplanted into the hindflank of the mice (8-10 mice per group, 2-10×10⁶cells/animal). Tumor growth is measured over 3 to 6 weeks using veniercalipers. Tumor volumes are calculated as a product of length x width xheight unless otherwise indicated. P values are calculated using theStudents t-test. Test compounds in 50-100 μL excipient (DMSO, orVPD:D5W) can be delivered by IP injection at different concentrationsgenerally starting at day one after implantation.

Tumor Invasion Model

The following tumor invasion model has been developed and may be usedfor the evaluation of therapeutic value and efficacy of the compoundsidentified to selectively inhibit KDR/FLK-1 receptor.

Procedure

8 week old nude mice (female) (Simonsen Inc.) are used as experimentalanimals. Implantation of tumor cells can be performed in a laminar flowhood. For anesthesia, Xylazine/Ketamine Cocktail (100 mg/kg ketamine and5 mg/kg Xylazine) are administered intraperitoneally. A midline incisionis done to expose the abdominal cavity (approximately 1.5 cm in length)to inject 10⁷ tumor cells in a volume of 100 μl medium. The cells areinjected either into the duodenal lobe of the pancreas or under theserosa of the colon. The peritoneum and muscles are closed with a 6-0silk continuous suture and the skin is closed by using wound clips.Animals are observed daily.

Analysis

After 2-6 weeks, depending on gross observations of the animals, themice are sacrificed, and the local tumor metastases to various organs(lung, liver, brain, stomach, spleen, heart, muscle) are excised andanalyzed (measurement of tumor size, grade of invasion, immunochemistry,in situ hybridization determination, etc.).

Additional assays

Additional assays which may be used to evaluate the compounds of thisinvention include, without limitation, a bio-flk-1 assay, an EGFreceptor-HER2 chimeric receptor assay in whole cells, a bio-src assay, abio-lck assay and an assay measuring the phosphorylation function ofraf. The protocols for each of these assays may be found in U.S.application Ser. No. 09/099,842, which is incorporated by reference,including any drawings, herein. Additionally, U.S. Pat. No. 5,792,783,filed Jun. 5, 1996 and U.S. application Ser. No. 09/322,297, filed May28, 1999 are incorporated by reference as if fully set forth herein.

Measurement of Cell Toxicity

Therapeutic compounds should be more potent in inhibiting receptortyrosine kinase activity than in exerting a cytotoxic effect. A measureof the effectiveness and cell toxicity of a compound can be obtained bydetermining the therapeutic index, i.e., IC₅₀/LD₅₀. IC₅₀, the doserequired to achieve 50% inhibition, can be measured using standardtechniques such as those described herein. LD₅₀, the dosage whichresults in 50% toxicity, can also be measured by standard techniques aswell (Mossman, 1983, J. Immunol. Methods, 65:55-63), by measuring theamount of LDH released (Korzeniewski and Callewaert, 1983, J. Immunol.Methods, 64:313, Decker and Lohmann-Matthes, 1988, J. Immunol. Methods,115:61), or by measuring the lethal dose in animal models. Compoundswith a large therapeutic index are preferred. The therapeutic indexshould be greater than 2, preferably at least 10, more preferably atleast 50.

Plasma Stability Test

The prodrug(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-onewas administered IV at 2 mg/mL to dogs. Levels of both prodrug and drug(3(Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1,3-dihydro-2H-indol-2-one)were followed by HPLC analysis of blood plasma for 4 hours followingdosing. This study showed that the half-life for conversion of theprodrug to drug was 7.3 min. From a plot of drug concentration vs time,the area under the curve indicated that 80% of the prodrug was convertedinto drug.

Formulation Examples

The formulations being evaluated are listed in Tables 1 and 2 and aredescribed below:

[A] Solid Formulations to be Reconstituted to a Stable Infusate (Table1):

(1) Lyophilized Formulation:

(a) Captisol Based: This formulation uses Captisol and an acidic agentto form an in situ salt at a pH of 1.5-2.0 to compound and lyophilizesolutions of drug at concentrations of 20.0-25.0 mg/mL. The lyophilizedcake is reconstituted with an IV fluid to provide a stable infusate at 2mg/mL or higher at pH 3.

(b) Non-C atisol based: This formulation uses small amounts of asurfactant such as Polysorbate-80 or Cremophor EL and an acidic agent toform an in-situ salt at a pH of 1.5-2.0 to compound and lyophilizesolutions of drug at concentrations of 20.0-25.0 mg/mL. The lyophilizedcake is reconstituted with cosolvent-surfactant based aqueous diluentsuch as PEG-300-Polysorbate 80 or PEG-300-Cremophor EL to provide astable infusate at 2 mg/mL or higher at pH 3.

(2) Sterile API Fill:

The drug is filled as a sterile powder fill in a container and will bereconstituted with a specific co-solvent—surfactant based aqueousdiluent to provide a stable infusate at 2 mg/mL or higher of drug at pH3.

[B] Solution Concentrate to be Diluted to a Stable Infusate (Table 2):

The drug is solubilized in a non-aqueous mixture of co-solvents andsurfactants at a high concentration such that it can be diluted withaqueous diluents to a stable infusate. The concentration of the drug inthe infusate is at a concentration of 2 mg/mL or higher, at pH 3. Thetotal levels of the co-solvent is less than 15% and the levels ofsurfactant is than 0.5%.

TABLE 1 Formulation (1) Solid formulations AttributesLyophilized-Captisol based Lyophilized Non-Captisol based Sterile APIFill Dose/50 CC 200-300 200-300 300-400 vial (mgs) Sterile API fill NANA 300-400 mg in vial Composition- drug (mg) 200-300 drug (mg) 200-300NA Lyophilized Acid (M) 1.4 Acid (M) 1.4 Cake Antioxidant (mg) 0-10Antioxidant (mg) 0-10 Captisol (mg) 2000-3000 Filler (mg) 200-300Polysorbate-80 (mg) 0-50 Composition- 0.9% NaCl, D5W, PEG-300 (% w/v)5-20 PEG-300 (% w/v) 5-20 Reconstitution Polysorbate-80 (% w/v) 0-1.0Polysorbate-80 (% w/v) 0-1.0 Fluid Citrate Buffer pH 3.0 Citrate BufferpH 3.0 0.1M (% w/v) 30-40 0.1M (% w/v) 30-40 Water (qs to volume) Water(qs to volume) Composition- drug (mg/mL) 2-3 drug (mg/mL) 2-3 drug(mg/mL) 2-3 Reconstituted Acid (Molar) 1.4 Acid (Molar) 1.4 InfusateAcid (Molar) 1.4 Antioxidant (mgmL) 0-1.0 Antioxidant (mg/mL) 0-1.0(Administered Filler (mg/mL) 2-3 Filler (mg/mL) 2-3 to Patient)Antioxidant (mg/mL) 0-1.0 PEG-300 (mg/mL) 50-200 PEG-300 (mg/mL) 50-200Polysorbate-80 (mg/mL) 0-10 Polysorbate-80 (mg/mL) 0-10 Captisol (mg/mL)20-30 Citrate Buffer 0.3 Citrate Buffer 0.3 0.1M, pH 3.0 (mL) 0.1M, pH3.0 (mL) IV fluid (qs to volume) Water (qs to volume) Water (qs tovolume) pH 3.0 pH 3.0 pH 3.0 Composition- drug (mg/mL) 20-30 drug(mg/mL) 20-30 NA Prelyophilate Acid (Molar) 1.4 Acid (Molar) 1.4Antioxidant (mg/mL) 0-10 Filler (mg/mL) 20-30 Antioxidant (mg/mL) 0-10Polysorbate-80 (mg) 0-50 Water for Injection qs to 1.0 mL Captisol(mg/mL) 200-300 pH 1.5-2.0 Water for Injection qs to 1.0 mL pH 1.5-2.0Acids used: Methane sulfonic acid, Tartaric acid, Citric acid, succinicacid is used in a 1:1.4 molar ratio for in situ salt formationCosolvents - PEG-300, PEG-400 Surfactants: Polysorbate-80, Cremophor ELCaptisol ®: Sulfobutylether Cyclodextrin Drug: Compound of Example 2

TABLE 2 Composition of Solution Formulation Composition (% w/v) orIngredients mg/mL Drug 1.2-2.5 (12-25 mg/mL) Cosolvent - (PEG-300, PEG-70-90 400) Surfactant 0-10 Dimethylacetamide 0-2 Salt forming agent(Methane Equivalent to 1.4M ratio of sulfonic acid, Tartaric acid, theAPI Citric Acid, Succinic Acid) Alcohol Qs to Volume

Formulation to be diluted 10 fold with pharmaceutically acceptable IVfluids.

The present invention is not to be limited in scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention. Indeed, various modifications of the invention in addition tothose described herein wail become apparent to those skilled in the artfrom the foregoing description. Such modifications are intended to fallwithin the scope of the appended claims.

All references cited herein are hereby incorporated by reference intheir entirety.

What is claimed is:
 1. A compound of the Formula (I):

wherein: R³, R⁴, R⁵ and R⁶ are independently selected from the group consisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, trihalomethane-sulfonamido, carbonyl, C-carboxy, O-carboxy, C-amido, N-amido, cyano, nitro, halo, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, amino and —NR¹¹R¹² where R¹¹ and R¹² are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, acetyl, sulfonyl, and trifluoromethanesulfonyl, or R¹¹ and R¹², together with the nitrogen atom to which they are attached, combine to form a five- or six-member heteroalicyclic ring provided that at least two of R³, R⁴, R⁵ and R⁶ are hydrogen; or R³ and R⁴, R⁴ and R⁵, or R⁵ and R⁶ may combine to form a six-membered aryl ring, a methylenedioxy group or an ethylenedioxy group; R⁷ is selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, carbonyl, acetyl, C-amido, C-thioamido, amidino, C-carboxy, O-carboxy, sulfonyl and trihalomethane-sulfonyl; R⁸, R⁹ and R¹⁰ are independently selected from the group consisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, carbonyl, C-carboxy, O-carboxy, cyano, nitro, halo, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino and —NR¹¹R¹², wherein R¹¹ and R¹² are as defined above; R^(1′) is hydrogen or alkyl; and R^(3′) and R^(4′) form an unsubstituted pyrrolidin-1-yl ring; or a pharmaceutically acceptable salt thereof.
 2. The compound of claim 1 wherein R^(1′) and R⁷ are hydrogen.
 3. The compound of claim 1, wherein R³, R⁴, R⁵, R⁶, R⁷, and R⁹ are hydrogen, and R⁸ and R¹⁰ are unsubstituted lower alkyl.
 4. The compound of claim 3, wherein R⁸ and R¹⁰ are methyl and R^(1′) is hydrogen.
 5. The compound of claim 1, wherein R³, R⁴, R⁵, R⁶, and R⁷ are hydrogen and R⁸ and R¹⁰ are unsubstituted lower alkyl.
 6. The compound of claim 5, wherein R⁹ is C-amido or lower alkyl substituted with carboxy and R^(1′) and R⁷ are hydrogen.
 7. The compound of claim 6, wherein R⁸ and R¹⁰ are methyl.
 8. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a compound of claim
 1. 9. The pharmaceutical composition of claim 8, wherein said composition is administered parenterally. 